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This study was aimed to investigate the changes of CD34(+) and CD71(+)CD45(-) cell levels in MDS and AA patients. A total of 25 cases MDS and 43 cases of AA (18 cases SAA and 25 cases of NSAA) from January 2010 to October 2013 in the Department of Hematology, affiliated hospital of Hebei United University were enrolled in this study. The complete blood count, bone marrow smears, bone marrow biopsy, karyotype analysis and bone marrow blood cell immune genotyping (mainly the proportion of CD34(+) cells, CD71(+)CD45(-) cells in nucleated cells) were carried out for all patients; the changes of CD34(+) and CD71(+)CD45(-) cell levels in patients with MDS and AA (SAA NSAA) were compared; the differences of white blood cell count, platelet count and hemoglobin concentration in patients with count of CD71(+)CD45(-) ≥ 15% or <15% were analyzed. The results showed that the count of CD34(+) in MDS group was higher than that in AA (NSAA and SAA) group (P < 0.05). The count of CD71(+)CD45(-) cells in MDS group was higher than that in SAA (P < 0.05), there was no significant difference between NSAA group and MDS group. In MDS group with CD71(+)CD45(-) ≥ 15%, the platelet count was significantly higher than that in NSAA group (P < 0.05); and there was no statistical difference for leukocyte, platelet count and hemoglobin level between MDS and NSAA group with CD71(+)CD45(-) <15% (P > 0.05). It is concluded that the count of CD34(+) cells in MDS patients is significantly higher than that in AA and SAA patients. The count of CD71(+)CD45(-) cells in MDS group is significantly higher than that of SAA group. The platelet count in MDS patients with CD71(+)CD45(-) cells ≥ 15% is significantly higher than that of the NSAA group.

Puma (P53 upregulated modulator of apoptosis) is a BCL-2 homology 3 (BH3)-only BCL-1 family member and a critical mediator of P53-dependent and -independent apoptosis. Puma plays an essential role in the apoptosis of hematopoietic stem cells exposed to irradiation without an increased risk of malignancies. This study was purposed to develop an effective lentiviral vector to target Puma in human hematopoietic cells and to investigate the effect of Puma gene knockdown on the biological function of human cord blood CD34(+) cells. SF-LV-shPuma-EGFP and control vectors were constructed, and packaged with the pSPAX2/pMD2.G packaging plasmids via 293T cells to produce pseudo-type lentiviruses. SF-LV-shPuma-EGFP or control lentiviruses were harvested within 72 hours after transfection and then were used to transduce human cord blood CD34(+) cells. GFP(+) transduced cells were sorted by flow cytometry (FCM) for subsequent studies. Semi-quantitative real time RT PCR, Western blot, FCM with Annexin V-PE/7-AAD double staining, Ki67 staining, colony forming cell assay (CFC), CCK-8 assay and BrdU incorporation were performed to determine the expression of Puma and its effect on the cord blood CD34(+) cells. The results showed that Puma was significantly knocked down in cord blood CD34(+) cells and the low expression of Puma conferred a radio-protective effect on the cord blood CD34(+) cells. This effect was achieved through reduced apoptosis and sustained quiescence after irradiation due to Puma knockdown. It is concluded that knockdown of puma gene in CD34(+) hematopoietic stem cells of human cord blood possesses the radioprotective effect, maintains the cells in silence targeting Puma in human hematopoietic cells may have a similar effect with that on mouse hematopoietic cells as previously shown, and our lentiviral targeting system for Puma provides a valuable tool for future translational studies with human cells.

This study was purposed to investigate the difference of nucleated cell (NC) count, CD34(+) cell ratio and expansion multiple, cell cycle and colony formation capability in in vitro expanded human umbilical cord blood CD34(+) cells from HOXB4-transfecting directly and HOXB4-transfected human umbilical cord mesenchymal stem cells (HUCMSC) by means of prepared feeder layers of HUCMSC. The HUCMSC were divided into 2 groups:first group, in which HOXB4 gene was transfected into HUCMSC by using lentiviral vecfor, and feeder layers were set up; and second group in which feeder layers for HUCMSC of non-transfected HOXB4 gene were set up. The CD34(+) cells were separated from HUCB by magmatic activated cell sorting(MACS). After culture in medium with cytokines for 2 days, CD34(+) cells were divided into 5 groups, including control group and experimental group. The control groups included CD34(+) cells as group A (blank control group) and GFP-CD34(+) cells as group B (negative control group) and experimental groups included HOXB4-CD34(+) cells as group C, HUCMSC+CD34(+) cells as group D, HOXB4-HUCMSC+ CD34(+) cells as group E and cells in all groups were cultured in vitro. The number of nucleated cells were counted at day 6, 10, 14 of culture and CD34 immunophenotypes, cell cycle and colony forming capability were measured at day 10 of culture in different conditions. The results indicated that HOXB4 gene could be transfected into HUCMSC by lentiviral vector and feeder layers were set up successfully. After culture for 14 days, the nucleated cells in 5 groups could be amplified effectively, and the expansion levels in 5 groups were in order HOXB4-HUCMSC+CD34(+) cell group> HOXB4-CD34(+) cell group>HUCMSC+CD34(+) cell group> control groups (P < 0.05). At day 10 of in vitro expansion the CD34(+) cell percentage decreased significantly in all groups, while the number of CD34(+) cell increased in experiment groups, which were in order HOXB4-CD34(+) cells group> HOXB4-HUCMSC+CD34(+) cell group>HUCMSC+CD34(+) cell group>control groups (P < 0.05). The cell cycle detection showed that the percentage of cells in S+G2/M phase in experiment groups were higher than that in control groups (P < 0.05), and percentage of cells in HOXB4-HUCMSC+CD34(+) cells group was higher (41.57%) than that in HOXB4-CD34(+) cells group(37.87%) and HUCMSC+CD34(+) cell group (28.65%) (P < 0.05). There was no statistical difference in the CFU number between HOXB4-HUCMSC+CD34(+) cell group and HOXB4-CD34(+) cell group, which were both higher than that in HUCMSC+CD34(+) cell group and control groups (P < 0.05).It is concluded that the CD34(+) cells cultured on HOXB4-HUCMSC feeder layers can be amplified significantly and kept the characteristics of stem cells, The feeder lager of HOXB4-HUCMSC is relative safe for amplification of CD34(+) cells in vitro, it possesses the potential useful value.

This study was aimed to investigate the feasibility of flow cytometry-fluorescence in situ hybridization (Flow-FISH) in measuring the telomere length of bone marrow cell subgroups in patients with myelodysplastic syndrome (MDS). Seven newly diagnosed patients with low-risk MDS and seven nutritional anemia patients who were matched with age and sex, were enrolled in this study. Heparinized bone marrow were sampled. Taking Molt-4 cell line as internal control cells, leukocytes isolated from whole bone marrow were labeled with CD34-Alexa Fluork ® 647, then denatured by high temperature and hybridized with FITC-conjugated telomere probe. The DNA was counterstained and the relative telomere length (RTL) of nucleated cells and CD34(+) cells in bone marrow were measured by four-color flow cytometry. The results showed that CD34(+) cells could be gated for the measurement of RTL in both groups, undergoing the denaturation and hybridization. Primary analysis indicated that the RTL of bone marrow CD34(+) cells in MDS patients was significantly shorter than that of bone marrow nucleated cells (P = 0.001), and the RTL of both CD34(+) cells and nucleated cells in bone marrow of MDS patients were significantly shorter than that of control group (P = 0.020, 0.002). It is concluded that the application of Flow-FISH in the measurement of RTL of certain cell subgroup is feasible by labeling the cell with thermostable fluorescence-conjugated antibody, and this technique is worthy to be investigated further.

This study was to establish the episomal vector reprogramming method to reprogram iPSC from human cord blood (CB) CD34(+) cells. The non-integrating plasmids of pEB-C5 and pEB-Tg were transfected into short-term cultured CB CD34(+) cells by using the nucleofector, so as to demonstrate efficient reprogramming of CB CD34(+) cells. Within 14 days of one-time transfection by two plasmids together, up to 200 iPSC-like colonies per 2 million transfected CB CD34(+) cells were generated. The results showed that the pluripotency of iPSC-derived CB CD34(+) cells was similar to that of hESC and the karyotypes of iPSC were normal. In addition, no vector integration was found in iPSC of 9th and 10th passages. Furthermore, hiPSC formed teratoma with three embryonic germ layers. It is concluded that the integration-free method to generate human iPSC from CB CD34(+) cells is reliable and can provide new ways for both research and future clinical applications.

Objective: To screen aptamers binding CD33+/CD34- cells from patients with acute myeloblastic leukemia M2 subtype (AML-M2)., Methods: CD33+/CD34- cells from patients with AML-M2 were taken as targeted cells, CD33+/ CD34- cells from normal people were taken as anti-selecting cells, and aptamers in the single strand deoxyribonucleic acid (ssDNA) library were then selected repeatedly by cell-systematic evolution of ligands by exponential enrichment (C-SELEX) technology, and amplified by polymerase chain reaction (PCR) to generate sub-ssDNA library. During the experiment, PCR amplification with fluorescently labeled primer and flow cytometry were performed to analyze the aptamers'enrichment of sub-library, and the final round product of the sub-ssDNA library was cloned. After the sequencing, the primary and secondary structures of the aptamers were analyzed., Results: Electrophoresis indicated that the product of PCR amplification for each round subssDNA library was able to see a clear DNA band in the agarose gel. After 13 rounds of screening, the fluorescence intensity of the sub-ssDNA library binding the cells ranged from 2.14% to 51.12%, reaching a steady state at the 13th round. A total of 30 clones were selected and sequenced, 22 of which contained 1 of the 4 conserved sequences of AAGTA, TATCT, AGATG and AAATT in their primary structure, but the remained eight aptamers contained none of the conserved sequence. Secondary structure analysis indicated that four stem-loops and loop simulation convex structures existed in the aptamers., Conclusion: C-SELEX technology can be used to screen the aptamers binding primary cells from patients with leukemia. The aptamers selected from the CD33+/CD34- cells from the patients of AML-M2 subtype might be used for the diagnosis and treatment for leukemia.

This study was purpose to investigate the biological characteristics of B lymphoblastic leukemia (B-ALL) between CD34 positive CD38 positive (CD34(+)CD38(+)) and CD34(+)CD38(low/-) subgroups and their clinical significance. Immunophenotyping of B cells in bone marrow of 54 patients with newly diagnosed CD34(+)B-ALL were analyzed by 4 color multiparametric flow cytometry (FCM). According to the different expression of CD38, the newly diagnosed patients with B-ALL were divided into two groups: CD34(+)CD38(+) subgroup and CD34(+)CD38(low/-) subgroup. BCR-ABL, TEL-AML1 fusion genes and WT1 gene were detected by real time RT-PCR simultaneously. After chemotherapy, minimal residual disease (MRD) was monitored by one tube of 7 color FCM. The average follow-up time was 12 months (range 1 - 28), the average follow-up interval was 2 months (range 1 - 5). The results showed that there was no significant differences such as WBC, Plt count and Hb level between the two groups at diagnosis, the positive rate of BCR-ABL, TEL-AML1 and WT1 gene was also no significantly different. After clinical complete remission (CR), MRD positive (MDR(+)) case rates were 28.57% (10/35) in CD34(+)CD38(+) subgroup and 68.42% (13/19) in CD34(+)CD38(low/-) subgroup (P < 0.01). The relapse rate between the two groups was 5.71% (2/35) in CD34(+)CD38(+) subgroup (relapse time at 94 and 245 d respectively) and 36.84% (7/19) in CD34(+)CD38(low/-) group [median relapse time was 263 d (range 46 - 468), P < 0.01]. The age distribution was analyzed in these two subgroups (> 16 or ≤ 16 years old), there was 8 (8/35) adult patients (> 16 years old) in CD34(+)CD38(+)group and 10 (10/19) adult patients in CD34(+)CD38(low/-) group (P < 0.05). It is concluded that CD34(+)CD38(low/-) phenotype is more often presented in adult patients and the CD34(+)CD38(low/-) patients with B-ALL are more likely to have MRD(+)and relapse after treatment.

Objective: To investigate the impact of the stents coated with sirolimus and anti-CD34 antibody on the short-term re-endothelialization and the long-term restenosis in Chinese Minipigs., Methods: Three different types of stents [bare-metal stent (BMS), sirolimus-eluting stent (SES) and anti-CD34 antibody and sirolimus-coated stent (ASES)] were randomly implanted in the coronary arteries of 22 Chinese Minipigs. At two weeks after stenting, coronary angiography and optical coherence tomography (OCT) were performed in 10 experimental animals. At three months after stenting, coronary angiography and OCT were performed in the remaining 12 experimental animals. Histopathologic examination was performed on the coronary artery segments containing stent after the animals were executed., Results: (1) No in-stent thrombosis and parietal thrombus were found by coronary angiography, OCT and histopathologic examination at two weeks post stenting. OCT analysis showed that the covered ratio of stent struts by neointima in ASES group was higher than in SES group [(55.56 ± 35.27)% vs. (41.82 ± 23.28)%, P < 0.05]. The mean thickness of neointima in ASES group was significantly higher than in SES group [(89.0 ± 5.0) µm vs. (32.0 ± 4.9) µm, P < 0.01] and BMS group [(89.0 ± 5.0) µm vs. (44.0 ± 7.2) µm, P < 0.01]. Histopathologic and scanning electron microscopy examinations demonstrated that the covering level and quality of stent struts by neointima in BMS and ASES group were both better than in SES group. (2) At three months follow-up, quantitative coronary angiography analysis found that late in-stent lumen loss in ASES group was significantly lower than in BMS group [(0.18 ± 0.06) mm vs.(0.35 ± 0.06) mm, P < 0.05]. OCT analysis showed that the percent neointimal hyperplasia in ASES and SES group was significantly lower than in BMS group [(34.75 ± 2.64)% and (35.63 ± 2.07)% vs. (48.28 ± 3.25)%, both P < 0.01]. Histopathologic analysis demonstrated that the percent areal restenosis of ASES and SES group were both significantly lower than that of BMS group [(28.65 ± 5.64)% and (29.33 ± 6.07)% vs. (46.18 ± 8.25)%, both P < 0.05]., Conclusion: The stents coated with anti-CD34 antibody and sirolimus can attenuate the inhibitory effect of sirolimus on the re-endothelialization at two weeks after stenting and the anti-hyperplasia effect of sirolimus at three months after stenting.

In order to investigate the influence of cytokine combinations on proliferation and differentiation of human umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro, the CD34(+) cells from human umbilical cord blood were amplified in serum-free medium StemSpan(SFEM) supplemented with several cytokine combinations by three-phase culture system. The effects of the cytokine combinations were compared. The results showed that at day 14 of the first culture phase, the CD34(+) cells cultured with cytokine combinations SCF + TPO + FL + IL-3 were amplified (11 000 ± 1 000) times, which were significantly higher than that of cells cultured with SCF + TPO + FL, but were not significantly different from that of cells cultured with SCF + TPO + IL-3 or SCF + TPO + FL + IL-3+ hydroxyl-corticosteroids. At day 7 of the second culture phase, the CD34(+) cells cultured with cytokine combination SCF + TPO + FL + IL-11 were amplified by (204666.7 ± 11718.9) times, which were significantly higher than that of cells cultured with SCF + TPO + FL + IL-3, but were not significantly different from that of cells cultured with SCF + TPO + FL + IL-11 + BMP4 + VEGF. At day 3 and day 6, the CD34(+) platelet-like cells accounted for about (39.8 ± 1.9)%, (39.7 ± 2.6)% and (25.5 ± 1.4)%, (23.1 ± 3.5)% cultured with SCF + TPO + FL + IL-11 and SCF + TPO + FL + IL-11 + BMP4 + VEGF, and significantly higher than that of the cells cultured with SCF + TPO + FL + IL-3. It is concluded that the cytokine combination of SCF + TPO + FL + IL-3 is most suitable cytokines combination for the amplification of CD34(+) hematopoietic progenitor cells. The cytokine combination of SCF + TPO + FL + IL-11 is preferred for the proliferation and differentiation of megakaryocytes, this study lays an experimental basis for investigating the proliferation and differentiation of CD34(+) into megakaryocytes/platelets in vitro.

A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment (cSELEX) was performed to screen the aptamers binding to CD33(+)/CD34(+) cells from the patients with acute myeloblastic leukemia (AML) of M(2) subtype (AML-M₂) so as to provide the basis for finding the specific marker on the surface of AML-M(2) CD33(+)/CD34(+) cells. Firstly, AML-M₂ CD33(+)/CD34(+) cells were sorted and used as targeted cells, and normal CD33(+)/CD34(+)cells were used as counter-targeted cells; the aptamers binding to CD33(+)/CD34(+) cells from patients with AML-M₂ were screened from the single strand deoxyribonucleic acid (ssDNA) library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M(2) CD33(+)/CD34(+) cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M₂ CD33(+)/CD34(+) cells, and the molecular diagnosis of the AML-M₂ leukemia.

Aim: To establish the transgenic cell strains expressing recombinant adenovirus vector of human Oncostain M(hOSM)gene which is supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34(+) hematopoietic stem/progenitor cell (HSPC) and compare its migration capacity before and after amplification in vitro., Methods: Establish the transgenic cell strains expressing recombinant adenovirus vector of hOSM gene, and the objective gene was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34(+) HSPC separated by magnetic-activated cell sorting (MACS) was detected by the FCM. After culturing with feeder layer cells, detect the rate of proliferation by flow cytometry (FCM). To compare the homing ability of HSPC after amplification in vitro, detect the spontaneous migration rate and migration rate induced by SDF-1 using transmembrane migration assay (Transwell experiment)., Results: The green fluorescence was observed by fluorescence microscope in the transgenic cell strains, and the objective gene was confirmed by RT-PCR and ELISA.The purity of umbilical cord blood CD34(+) HSPC separated by MACS could reach(96.8 ± 2.28)%. After culturing with feeder layer cells for 7 days, the CD34(+) cells were 15.73 times in group containing hOSM more than in group without hOSM. The expression rate of adhension molecules on the surface of CD34(+) cells were also higher in the group containing hOSM than without hOSM. After using Transwell assys to detect the homing ability of culturing cells, the induction migration rate of stem cells clturing on transgenic cell strains was (40.68 ± 1.35)%, significantly higher than the control, which reveals a better homing ability., Conclusion: Recombinant adenovirus vector of hOSM gene as feeder layer cells can effectively proliferate umbilical cord blood CD34(+) HSPC in vitro and delay it differentiate, what's more, the stem cells retain a high homing ability after culturing on transgenic cell strains in vitro.

Objectives: To detect the abnormalities of CD(34)(+) cells differentiation and bone marrow cell cycle in myelodysplastic syndrome (MDS)., Methods: Fifty newly diagnosed MDS (17 in low risk and 33 in high risk), 8 acute myeloid leukemia preceded by MDS (MDS-AML) and 25 normal controls were enrolled into this study. Their CD(34)(+)CD(38)(+), CD(34)(+)CD(38)(-) bone marrow cells and bone marrow cell cycle were measured with flow cytometry., Results: The mean percentages of CD(34)(+) cells in bone marrow karyocyte of high risk [(2.29 ± 2.17)%] and MDS-AML groups [(18.69 ± 17.47)%] were significantly higher than that of control group [(0.36 ± 0.49)%, P < 0.05]. The mean percentages of CD(34)(+)CD(38)(+) cells were significantly lower in low risk, high risk and MDS-AML groups [(86.09 ± 7.79)%, (81.68 ± 11.82)% and (82.88 ± 2.60)%, respectively] than that in control group [(92.21 ± 3.85)%, P < 0.05], thus the percentages of CD(34)(+)CD(38)(-) cells were significantly higher in either MDS (low risk and high risk) or MDS-AML groups [(13.91 ± 7.79)%, (18.32 ± 11.82)% or (17.13 ± 2.60)%, respectively] than that in control group [(7.79 ± 3.85)%, P < 0.05]. The percentages of CD(34)(+)CD(38)(-) cells of MDS cases correlated directly with their International Prognostic Scoring System (IPSS) (r = 0.493, P = 0.001) and WHO Adapted Prognostic Scoring System (WPSS) (r = 0.586, P = 0.000) scores. The percentages of bone marrow mononuclear cells (BMMNCs) in G(0)/G(1) phase of in low risk, high risk and MDS-AML groups [(94.52 ± 4.32)%, (96.07 ± 3.88)% and (94.65 ± 4.55)%, respectively] were significantly higher than that in control group [(88.94 ± 7.30)%, P < 0.01], thus the percentages of BMMNCs in S and G(2)/M phase were significantly lower in either MDS (low risk and high risk) or MDS-AML groups than that in control group (P < 0.05). MDS patients with low percentages of CD(34)(+)CD(38)(-) cells presented higher therapeutic efficacy than those with high percentages of CD(34)(+)CD(38)(-) cells, while without significant differences (P > 0.05)., Conclusions: There are abnormalities of differentiation of CD(34)(+) bone marrow cells and high proportion of G(0)/G(1) cells which indicates a G(1) phase arrest in MDS that might be involved in the pathogenesis of MDS. So the examination of CD(34)(+) bone marrow cells and cell cycle might be helpful for MDS diagnosis and assessment of prognosis and therapeutic effects.

The purpose of this study was to investigate the immunophenotyping characteristics of adult acute lymphoblastic leukemia (ALL) patients in groups of different ages. Immunophenotyping was performed in 260 ALL patients by flow cytometry using a panel of monoclonal antibodies and CD45/SSC gating. The results indicated that (1) all the 82 cases of T-cell acute lymphoblastic leukemia (T-ALL) expressed CD7 (100%) while the positive rate of CD2 remarkably decreased with aging. The positive rate of CD2 in patients aged 14 to 18 years (adolescents) was 91.67%, which is significantly higher than that in cases aged 19 to 35 years (young adults) and > 35 years (older adults) (65.71% and 43.48% respectively, p < 0.05); the positive rate of CD34 and HLA-DR increased with aging, there was significant difference of the HLA-DR expression between the older adults group (39.13%) and the other two groups (4.17% in adolescents and 11.43% in young adults respectively (p < 0.05). Moreover, there were significant differences of the myeloid antigen (MyAg) and CD13 expression between the older adults and younger adults (p < 0.05). (2) As to adult B-cell acute lymphoblastic leukemia (B-ALL), the positive rates of CD19 and HLA-DR in 178 cases were 100%; the positive rate of CD33 in young adults was significant higher than that in adolescents (p < 0.05), the differences of the other marker expressions failed to reach statistical significance in adult B-ALL patients. It is concluded that the immunophenotypes of adult T-ALL are evidently heterogeneous in different ages, and expression with more aberrant phenotypes indicates poor prognostic significance in patients older than 35 years. There is no significant association of immunophenotypes with ages among different age groups of adult B-ALL.

To investigate whether the RBC lysing solution can affect the results of relative enumeration of CD34(+) cells, 37 mobile peripheral blood apheresis products were stained using CD34-PE and CD45-FITC monoclone antibodies and RBCs were then lysed by two lysing solution commercially available (one named FACS Lysing Solution, FACS; another IOTest 3 Lysing Solution, IOTest) and one lysing solution self-prepared. After being processed by lyse-and-then-washed method, samples were detected by FACSC anto flow cytometer. The percentages of CD34(+) cells were determined based on ISHAGE gating strategy, forward and side scatter (FSC and SSC) characteristics, percentage of CD45(+) cells were recorded simultaneously. The results showed that by lyse-and-then-wash method, the percentages of CD34(+) cells in FACS-treated samples were significantly lower compared with that in IOTest-treated samples (0.50 +/- 0.42 vs 0.92 +/- 0.59, p = 0.004), but no statistical difference was observed between IOTest-treated and ourselves-prepared-treated samples. The intensities of FSC and SSC in cells of IOTest-treated sample were significantly higher compared with that in cells of FACS-treated sample (p < 0.01). The proportion of CD45(+) cells in IOTest-treated samples was lower than that in FACS-treated samples. The WBC count of samples was not correlated to the amount of CD34(+) cells (r(s) = 0.192, p = 0.357). It is concluded that the red cell lysing solution shows unexpected effect on detecting and counting CD34(+) cells, prudence should be taken to select such reagents at FCM performance.

In order to investigate the effect of anti-TGF-beta1 monoclonal antibody on the expansion of cord blood CD34(+) cells, the purified cord blood CD34(+) cells were divided into three groups: blank control group: purified cord blood CD34(+) cells cultured on day 0; control group: cells cultured for 3 days in the culture system, containing SCF, IL-3, IL-6 and FLT3-L; test group: cells cultured for 3 days in the same culture system as control group, but with anti-TGF-beta1 monoclonal antibody. The mononuclear cell counting (MNC), the expression of CD34 and c-kit, and CFU-GEMM, BFU-E and CFU-GM counting were detected in all three groups. The result showed that the expansion of MNCs, CD34(+) cells and CD34(+)c-kit(+) cells in test group [(2.35 +/- 0.25) x 10(5), (1.16 +/- 0.29) x 10(5), (1.09 +/- 0.26) x 10(5)] was significantly higher than that in control group [(1.25 +/- 0.13) x 10(5), (0.55 +/- 0.19) x 10(5), (0.51 +/- 0.2) x 10(5)](p < 0.01). The expansion of more primitive CD34(+)c-kit(-) subpopulation in test group [(12.95 +/- 3.17) x 10(3)] was even significantly higher than in control group (1.71 +/- 0.83) x 10(3) (p < 0.01). Colony forming assay showed that the number of earlier progenitor colony CFU-GEMM, BFU-E in test group [(16.3 +/- 4.72) x 10(3), (60.5 +/- 20.96) x 10(3)] was higher than that in control group [(5.0 +/- 2.58) x 10(3), (16.25 +/- 7.93) x 10(3)] (p < 0.01). The number of relatively mature CFU-GM between test group and control group was not statistical significance [(6.33 +/- 2.85) x 10(3) vs (4.0 +/- 2.28) x 10(3)](p > 0.05), but both were higher than that in blank group (0.75 +/- 0.29) x 10(3). These results demonstrated that anti-TGF-beta1 monoclonal antibody promoted the expansion of MNC and CD34(+) cells, and even more marked expansion of the more primitive progenitor cells-CD34(+)c-kit(-) cells. Meanwhile, it enhanced the output of more immature colony CFU-GEMM and BFU-E, but had no evident influence on the mature myeloid colony CFU-GM. It is concluded that the anti-TGF-beta1 monoclonal antibody can synergize other cytokines to enhance the proliferation of cord blood CD34(+) progenitor cells effectively, and it is more important that can reserve more primitive progenitor cells.

This study was aimed to explore the effect of donor characteristics (age, sex and so on.) on CD34(+) cell yields in apheresis from healthy donors mobilized by recombinant granulocyte colony-stimulating factor(rhG-CSF). In 61 healthy donors, the characteristics associated with CD34(+) cell yield were analysed. The relationship between the CD34(+) cell yields and donor characteristics was statistically assessed with multivariate forward, backward and stepwise regression methods. A variety of parameters were analyzed which included donor age, sex, weight, height, body mass index (BMI) and time for collection of peripheral blood apheresis, while the mean number of peripheral blood mononuclear cells (MNCs), CD34(+) cell count, CD34(+) cell proportion based on MNC and CD34(+) cell count per kg of donor weight were used as the variables. The results showed that age of donors was the main factor impacting CD34(+) cell yields (-0.60 < r < -0.45, p < 0.005). In a partial correlation analysis the body weight, height and BMI were served as control factors, the negative correlation of age with CD34(+) cell yields was still found (-0.50 < r < -0.35, p < 0.02). BMI was only weakly correlated with the yields of CD34(+) cells per kg(r = -0.297, p < 0.05). As a whole, sex showed no relation with the CD34(+) cell yields. Compared with the female group less than 35 years old, height, weight and BMI in male group of low age exerted a positive impact on CD34(+) cell yield. The optimal time for collection of PB was day 4 after treatment with rhG-CSF, when 70% of the donors could reach the peak CD34(+) cell yields. It is concluded that the age of the donors is the first factor determining the choice of donors for allogeneic hematopoietic stem cell transplantation, the sex, height, weight and BMI are secondary factors impacting yield of CD34(+) cells from donors mobilized with rhG-CSF.

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. The expressions of JunB and CDH13 (cadherin-13) gene as tumor suppressor gene were inactivated by promoter methylation in CML patients. This study was purposed to investigate the methylation difference of JunB and CDH13 gene promoter and the expression levels of JunB and CDH13 gene in CD34(+)CD38(-) cells in CML patients vs normal individuals. CD34(+)CD38(-) cells from 8 cases of CML and 5 normal individuals were selected by flow cytometry. The methylation status of JunB and CDH13 genes were detected by MS-PCR in selected CD34(+)CD38(-) cells. The expression levels of JunB and CDH13 gene was detected with real time polymerase chain reaction (RT-PCR). The results showed that no methylation of JunB and CDH13 gene was detected in CD34(+)CD38(-) cells of 5 normal individuals. Methylations of JunB and CDH13 promoter were found in 87.5% (7/8) and 50% (4/8) CML CD34(+)CD38(-) cells, percentages of which were significantly higher than those in normal individuals. The difference was statistically significant (p < 0.05). The relative expression levels of JunB and CDH13 mRNA in CD34(+)CD38(-) cells of CML patients were significantly lower than those in normal individuals (2(-DeltaDeltaCT) were 1/5.21 and 1/10.63 respectively). It is concluded that the high methylation of JunB and CDH13 gene promoter occurs in CD34(+)CD38(-) cells of CML patients, their mRNA expression level is significantly lower, thus the methylation of JunB and CDH13 gene promoter probably plays a role in the pathogenesis of CML and may have clinical significance in predicting prognosis of CML.

This study was purposed to clarify whether biology function of mesenchymal stem cells (MSCs) is changed by suppressing the development of dendritic cells (DC) derived from hematopoietic stem cells (HSCs). MSCs were cocultured with dendritic cells derived from CD34 positive hematopoietic stem cells (HSCs), and then the expression of cytokines and phenotypes of DCs/MSCs were detected by RT-PCR and flow cytometry respectively. Induced experiments were used to analyze the differentiation ability of MSCs. The results showed that DCs/MSCs were negative for the CD14, CD34, CD45, CD31, CD86, but positive for HLA-ABC, CD29, CD73, though the percentage decreased as MSCs vs DCs/MSCs (93.1% vs 13.44%, 98.3% vs 78.8%, 95.3% vs 75.9%). In addition, the expression of cytokines such as M-CSF, TGF-beta increased in DCs/MSCs. After differentiation induction, DCs/MSCs were deprived of the potential to differentiate into adipocytes, but maintained osteogenesis characteristics. It is concluded that the basic characteristics of MSCs are altered after coculture with DCs, and DCs/MSCs result in lower expression of mesenchymal phenotypes and decrease differentiation ability, but increase the expression of cytokines related to hematopoiesis and immunity.

Aim: To investigate the relationship between micro-lymphatic vessel density (MLVD) and lymphatic metastasis in follicular thyroid carcinoma (FTC)., Methods: MLVD and micro-vascular density (MVD) were determined and counted by Envision immunohistochemistry using D2-40 and CD34 monoclonal antibodies in 35 specimens of follicular thyroid carcinoma(FTC)and 20 of nodular goiter. The relationship between MLVD and lymphatic metastasis was analyzed., Results: MLVD and MVD in follicular thyroid carcinoma were closely related with lymphatic metastasis. At the same time, the cancer embolus in the micro-lymphatic vessel was significantly related with the lymphatic metastasis (P<0.01)., Conclusion: The metastasis process of follicular thyroid carcinoma (FTC) is associated with the formation of the micro-lymphatic vessel and micro-vascular. Inhibition of the lymphatics formation may be a potential therapeutic maneuver for thyroid neoplasms in the future.

This study was purposed to investigate the relation of monocyte counts in peripheral blood (PB) at the first collection of allograft to the amount of CD34(+) cells in the mixture of recombinant human granulocyte colony-stimulating factor (rhG-CSF)-primed bone marrow graft (G-BM) and rhG-CSF mobilized peripheral stem cell grafts (G-PB). 70 healthy donors were treated with rhG-CSF [5 microg/(kg.d)] injected subcutaneously for 5 consecutive days. Bone marrow grafts and peripheral blood grafts were harvested on the 4th and 5th days respectively. Blood cell counts at the first collection of allografts were determined by blood analyzer XL2100, the amount of CD34(+) cells in G-BM and G-PB were determined by flow cytometry. The results showed that the monocyte counts in the PB of the 70 healthy donors were (1.15 +/- 0.60) x 10(9)/L. The amount of total CD34(+) cells in the G-BM, G-PB and mixture grafts were (5.85 +/- 2.93) x 10(7), (1.33 +/- 0.77) x 10(8), and (1.92 +/- 0.86) x 10(8) respectively. Pearson and Spearman correlation analysis indicated that the counts of monocyte at the first collection of allografts correlated positively with the amount of total CD34(+) cells in the G-BM (correlation coefficient, r = 0.265, p = 0.027), G-PB (r = 0.340, p = 0.004) and mixture grafts (r = 0.398, p = 0.001). Stepwise Linear Regression Model analysis also showed that the counts of monocytes in the PB correlated positively with the amount of CD34(+) cells in the G-BM, G-PB and mixture grafts (p = 0.027, 0.004 and 0.001 respectively). The sensitivity and specificity of the monocyte counts to predict the amount of CD34(+) cells in the mixture grafts were 71% and 70% respectively (p = 0.007). In conclusion, the monocyte counts in the PB at the first collection of allografts after rhG-CSF treatment of healthy donors may be a simple and practical indicator for harvest of CD34(+) cell amount in the mixture grafts.

This study was aimed to investigate the immunophenotypic characteristics of acute promyelocytic leukemia (APL). CD45/Side Scatter (SSC) gating strategy and multiparametric flow cytometry were used to determine immunophenotype of 143 patients with APL. The immunophenotypic features were compared between newly diagnosed APL patients and relapsed APL patients. 42 patients with HLA-DR(-) (non-APL AML, DR(-)AML) were randomly selected as controls. 31 out of 42 AML patients were CD34 negative, and their immunophenotypes were compared with those in newly diagnosed APL patients. The results showed that (1) CD34 and HLA-DR were both negative in 91.9% of newly diagnosed APL, while the positive rate of CD34 and HLA-DR elevated in relapsed cases (3.0% vs 37.5%, 3.9% vs 37.5%). The positive rate of CD34 in HLA-DR(-) AML group was higher than that in newly diagnosed APL group (23.4% vs 3.0%). The positive level of CD34 in newly diagnosed APL group was lower than that in HLA-DR(-) AML group; (2) the positive rate of CD33 in newly diagnosed APL group was higher than that in other groups (97.0% vs 75.0%, 83.3%, 83.9%), as well as the the positive level of CD33 (p < 0.05). (3) no lymphoid antigen other than CD2 was expressed in newly diagnosed APL group. The positive rate of CD7 was 9.5% in DR(-) AML group and 6.5% in CD34(-)/DR(-) AML group, both were higher than those of newly diagnosed APL group (p < 0.05). It is concluded that the immunophenotyping can provide proof to the rapid diagnosis of APL. For those patients with DR(-) AML, it may be helpful to identify APL depending on following features: low or negative CD34 expression, homogeneous and bright expression of CD33, no lymphoid antigens other than CD2, higher SSC.

This study was purposed to investigate the correlation between the dose infused megakaryocytic precursors (CD34+, CD34+CD61+) and recovery time of platelet count following an allogeneic PBSCT and/or BMT through quantitative detection of CD34+ and its subpopulation in peripheral blood and BM mobilized by G-CSF. 24 patients with various hematologic malignancies received PBSCT/BMT from their HLA matched or unrelated donors and haploidentical siblings in April-December 2007. 20 evaluated patients were divided into 2 groups according to different transplant schemes. HLA matched group received PBSCT regime and haploidentical group received PBSCT combined with BMT. CD34+CD61+ subpopulations in sample from patients receiving PBSCT/BMT were measured by flow cytometry immediately or storage over night. The results showed that the median number of infused CD34+, CD34+CD61+ and CD34-CD61+ cells in haploidentical group were 6.24x10(6)/kg (1.53-20.48), 66.19x10(4)/kg (8.16-493.83), and 34.38x10(6)/kg (14.71-109.16) respectively, in HLA matched group those were 4.88x10(6)/kg (1.00-8.24), 14.16x10(4)/kg (11.63-96.87), and 13.50x10(6)/kg (1.74-35.61), respectively. Median days of ANCs>0.5x10(9)/L and platelets>20x10(9)/L were 18.5 (11.0-29.0) days and 16.5 (9.0-35.0) days in haploidentical group respectively; in HLA matched group those were 14.5 (9.0-24.0) and 10.5 (6.0-37.0) respectively. A significance difference of median days for ANC engraftment presented between two groups (p=0.048). There was no significant difference of time for platelet engraftment between 2 groups. For patients with CD34+ cell dose>2x10(6)/kg there was significant difference of time of platelet engraftment between HLA matched and haploidentical groups (p=0.006). The number of CD34+CD61+ cells infused in 12 haploidentical patients or in 8 HLA matched patients were much better correlated with the time of platelet recovery up to 20x10(9)/L than that of number of CD34+ cells infused in total 20 patients (r=-0.768 and p=0.004 for haploidentical CD34+CD61+ cells, r=-0.747 and p=0.033 for HLA matched CD34+ CD61+ cells, r=-0.449 and p=0.047 for CD34+ cells). There was an inverse correlation between the number of infused CD34+ CD61+ cells and time of platelet engraftment. Therefore, as the number of CD34+ CD61+ cells increased, duration of platelet engraftment (time to reach platelet count of 20x10(9)/L) shortened significantly. It is concluded that the determining the number of megakaryocytic precursor by flow cytometry may predict the platelet reconstitutive capacity of the allogeneic hematopoietic stem cell transplantation, which is in haploidentical PBSCT and in BMT.

This study was aimed to detect the ratio of CD34+ cells in bone marrow mononuclear cells (BMMNCs) and the expression rate of G(M)-CSFR on CD34+ cells in bone marrow of the patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). The ratio of CD34+ cells in BMMNCs and the expression rate of G(M)-CSFR on cells of 27 AA patients, 45 MDS patients and 20 controls were detected by flow cytometry (FCM). The results showed that the ratio of CD34+ cells in BMMNCs of AA patients reduced and was significantly different from controls (p<0.05), the ratio of CD34+ cells in MDS patients elevated and was significantly different from controls (p<0.05). Compared with controls and MDS-RA patients, the ratio of CD34+ cells in MDS-RAEB patients significantly elevated (p<0.05), but there was no significant difference between MDS-RA patients and controls (p>0.05). The ratio of CD34+ cells in MDS-RA patients was significantly higher than that in AA patients (p<0.05). There was no significant difference in expression rate of G-CSFR on CD34+ cells between AA patients and controls, MDS patients and controls, AA patients and MDS patients, MDS-RA patients and MDS-RAEB patients (p>0.05). The expression rate of GM-CSFR in MDS patients was significantly higher than that in AA patients and controls (p<0.05), but there was no significant difference between AA patients and controls, MDS-RA patients and MDS-RAEB patients (p>0.05). In AA patients, the ratio of CD34+ cells in BMMNCs was less than 0.1% accounts for 6/8 SAA patients, compared with 2/19 in CAA (p<0.05). There was no correlation between the expression rate of either G-CSFR or GM-CSFR and neutrophil count at diagnosis (r=0.058 and r=0.044). In MDS patients, there was no correlation between bone marrow CD34+ cells ratio and peripheral neutrophil count at diagnosis (r=-0.335). And there was no correlation between the expression of either G-CSFR or GM-CSFR and neutrophil count on diagnosis (r=0.064 and r=0.051). It is concluded the detection of CD34+ cells and their surface expression rate of G(M)-CSFR in AA and MDS is useful in diagnosis and differential diagnosis of these two diseases.

This study was purposed to investigate the biological characteristics and immunogenicity changes of ex vivo expanded megakaryocyte progenitors from human umbilical cord blood CD34(+) cells in order to provide experimental basis for clinical application of ex vivo expanded umbilical cord blood megakaryocyte progenitor cells. Mononuclear cells (MNCs) were obtained from umbilical cord blood by Ficoll-Hyapaque density gradient separation. CD34(+) cells were enriched by magnetic cell sorting (MACS). The selected CD34(+) cells were seeded in serum-free medium stimulated with thrombopoietin (TPO, 50 ng/ml), interleukin 11 (IL-11, 50 ng/ml), and heparin (25 U/ml) for 14 days. The immunophenotyping (CD34(+), CD41a(+), CD61(+), CD34(+) CD41a(+) and CD34(+) CD61(+) cells) of amplificated products, matured megakaryocyte apoptosis, and expression of human leukocyte antigen (HLA) class I and class II molecules were measured by fluorescence-activated cell sorter (FACS). The number of colony-forming units-megakaryocyte (CFU-Mk) was also evaluated by CFU-Mk assay. The results showed that the umbilical cord blood CD34(+) mononuclear cells could be effectively differentiated into megakaryocytes. The peak expression ratios of CD41a(+) and CD61(+) cells were all at 14th days, while that of CD34(+) CD41(+) and CD34(+) CD61(+) cells were at 7th day [(3.41 +/- 2.80)% and (1.89 +/- 1.43)%, respectively]. The expansion times of large and small CFU-Mk reached peak at 7th day (20.66 +/- 32.79) and 10th day (435.62 +/- 482.65), respectively. The apoptotic rates of megakaryocytes at 7th, 10th, 14th day were (19.48 +/- 9.64)%, (26.87 +/- 9.03)%, and (52.46 +/- 11.74)%, respectively. The apoptotic rate of megakaryocytes had no significant difference in 7 and 10 days culture (p > 0.05), while that significantly increased in culture for 14 day culture, compared with culture for 7 and 10 days (p < 0.05, respectively). The expression of HLA class I and class II molecules on megakaryocytes decreased along with the prolongation of expansion time and sharply decreased in 0 to 10 days. It is concluded that the cytokines of TPO, IL-11, and heparin can promote the expansion of megakaryocyte progenitors from umbilical cord blood CD34(+) mononuclear cells effectively in vitro. The peaked expansion times of large CFU-Mk, the peaked expression ratios of CD34(+) CD41(+) and CD34(+) CD61(+) cells were all at 7th day. So the culture for 7 days appeared to be the optimal duration of expanding megakaryocyte progenitors.

Aim: To study the hematopoietic reconstitution ability of fresh and cultured cord blood CD34(+) cells by sublethally irradiated NOD/SCID mice., Methods: Mononuclear cells (MNC) were separated from cord blood and then cultured with stem cell factor (SCF)+ flt3 ligand (FL)+ thrombopoietin (TPO)+ interleukin-3 (IL-3)+ IL-6 for 14 d. CD34(+) cells were isolated from fresh or cultured MNC using miniMACS magnetic separation system. Then 4x10(5) CD34(+) cells together with 5x10(6) CD34(-) cells were injected into NOD/SCID mice. After transplantation, the dynamics of hematopoieitc recovery in the mice were measured. After 6 weeks, bone marrow (BM) and spleen samples were obtained from mice for detecting human cells., Results: The number of total cells increased 1.78-fold after 14 d of culture. All recipients received fresh and cultured CD34(+) cells survived. Human cells, human lineage cells and human ALU sequence could be detected in BM and spleen. The peripheral blood counts of all transplanted mice recovered. The engraftment levels of cultured CD34(+) cells were similar to those of fresh CD34(+)cells but the percentage of human lineage cells was higher than that of fresh CD34(+) cells., Conclusion: CD34(+) cells cultured for 14 d still preserved the hematopoietic reconstitution ability and showed better multilineage reconstitution ability than fresh CD34(+) cells.

Rapid re-endothelialization of implanted coronary artery stent is a new way for preventing restenosis. In this paper, adhesive polypeptide mimics of marine organism and antibodies potentially selective targeting with CD34 antigens on endothelial cells (ECs) were immobilized onto ethylene-vinyl acetate copolymer (EVA) coatings for improving the re-endothelialization process. The results showed that the attachments, growths, viabilities, as well as cell retentions of ECs on EVA were improved after modification. The immobilized polypeptide promoted the attachments of ECs, but overmuch polypeptide would restrict the attachments. With the increase of antibody concentration, the immobilized antibodies and cell attachments were improved.

Aim: aTo study the effect of human nuclear distribution C èhNUDCé on human megakaryocyte proliferation and differentiation from cord blood CD34(+) cells in vitro., Methods: aHuman CD34(+) cells were isolated using the Dynal CD34 Progenitor Cell Selection System from umbilical cord blood. The CD34(+) cells were then cultured in serum free methylcellulose semi-solid media, the morphologic aspects and number of small, medium and large CFU-MK colonies were observed and scored on the day12 by microscopy analysis. The CD34(+) cells were cultured in serum free liquid media, cells were removed on day 10 and formation of CD41(+) in human megakaryocyte and its DNA polyploidization of nuclear were analyzed on a FACsort flowcytometer., Results: ahNUDC supported the formation of small and medium CFU-MK colony in serum free semi-solid media. Furthermore, hNUDC induced a remarkable increase in expression of the megakaryocyte cell surface marker CD41(+) and stimulated the CD41(+) DNA polyploidization more effectively than TPO., Conclusion: hNUDC may play an important role in megakaryocyte proliferation and differentiation.

Aim: To investigate the effects of adipose-derived stem cell (ADSC) transplantation on the angiogenesis in the brain post focal cerebral ischemia in rats., Methods: 72 male adult Sprague-Dawley rats were randomly divided into 4 groups: sham-operated group, middle cerebral artery occlusion (MCAO) group, vehicle group and MCAO+ADSC-treated group (n=18). A permenant focal cerebral ischemia model was established with the modified Longa's method. ADSC were labeled by DAPI before transplantation. One day after right MCAO, 30 muL of cell suspension containing 1x10(6) cells were injected into the lateral ventricle of MCAO+ADSC-treated group and the same dose of PBS was given to the vehicle group. On D4, D7 and D14 after MCAO, the rats were killed to detect the regeneration of microvessel and the expression of bFGF and VEGF in ischemic region by immunohistochemistry and RT-PCR., Results: A lot of microvessel proliferate in the injured cortex reached peak in 2 weeks. The microvessel density in the brain tissues of rats treated with ADSC was higher than that in MCAO group and vehicle group (P<0.01). The expression of bFGF and VEGF in the brain tissues of MCAO+ADSC-treated group was higher than that in MCAO group and vehicle group on D4, D7 and D14 post MCAO., Conclusion: The transplantation of ADSC can promote the revascularization of cerebral ischemia in rats partly by enhancing bFGF and VEGF synthesis in brain.

Depletion of T and B cells from the graft is prerequisite for haploidentical transplantation to decrease the risk of GVHD and EBV-associated lymphoproliferative disease. This study was aimed to investigate the performance of T-cell and B-cell simultaneous depletion from mobilized peripheral blood stem cells (PBSCs) for the first time in China, using anti-CD3 and anti-CD19 antibodies conjugated to magnetic microbeads by the CliniMACS device. The depletion efficiency of T-cell and B-cells was analyzed by flow cytometry; the function of the stem cells after depletion was evaluated using colony assays. The results indicated that the mononuclear cell count prior to T- and B-cell depletion was 4.88 x 10(10). After depletion, the percentage of T cells was 0.02% with a log (10) depletion of 4.4. The percentage of B cells was less than 0.01% with a log (10) depletion of at least 3.3. The product contained not only CD34(+) stem cells, but also NK cells, monocytes and granulocytes. After T- and B-cell depletion the purity of CD34(+) cells was 0.98%, the number of CD34 cells was 1.84 x 10(8) and their recovery rate was 69.7%. The number of NK cells was 2.54 x 10(9) and the recovery rate of NK cells was 71.7%. In vitro colony assays showed no negative impact on function of the hematopoietic stem cells. In conclusion, the CliniMACS system can be used to efficiently deplete T and B cells from PBSCs simultaneously, without adverse effect on biological function of hematopoietic stem cells. This study provides technical platform for haploidentical hematopoietic stem cell transplantation.

Objective: To investigate the variation of immune index in patients with systemic lupus erythematosus (SLE) treated with autologous purified CD34+ cells transplantation and to clarify the relationship with pathogenesis and prognosis., Methods: Flow cytometry (FCM) and enzyme linked immunosorbent assay (ELISA) were used to test lymphocyte subsets, C3, C4, CH50, autoantibodies and immunoglobulin for 18 cases of SLE before and after transplantation., Results: The results showed that the ratio of all the T cell subsets reduced obviously in early post graft and recovered gradually in 1 to 3 months after transplantation except CD45RO(+)CD4(+) cells. The levels of serum C3, C4, CH50 increased significantly after transplantation. No case relapsed within one year after transplantation, but 2 patients relapsed one year after transplantation. The levels of the indexes in the patients with relapse were significantly lower than those in the patients with persistent remission, including C(4) in the entire course, CH(50) in the 3rd and 12th month after transplantation and CD(45)RA(+)CD(8)+ cells in the 6th month after transplantation. However, the ratio of CD45RO(+)CD(4)+ cells in the first month after transplantation in the patients with relapse was higher than that in the patients with persistent remission., Conclusion: Autologous purified CD(34)+ cells transplantation is effective for treating SLE. Survey of immune indexes before and after transplantation is important to investigate the pathogenesis of SLE. Moreover, these immune indexes can be used to predict therapeutic efficacy of SLE.

To establish a basis for deep investigation of the role of microRNA (miRNA) in the regulation of hematopoiesis, differential expression profiles of miRNA between human cord blood CD34(+)CD38(-) and CD34(+)CD38(+) cells were analyzed. Mononuclear cells from cord blood (CB) of healthy donors were separated by Ficoll-Hypaque density gradients. CD34(+)CD38(-) and CD34(+)CD38(+) cells were sorted by using FACS Vantage SE. Their mRNA were then extracted and hybridized to miRNA microarray chip. The resulting data were analyzed with GeneSpring and informatics technique. The results showed that eleven miRNAs were found to be downregulated and 73 miRNAs to be upregulated by at least two-fold in the CD34(+)CD38(+) cells of CB, compared with the CD34(+)CD38(-) cells, which maintained CD34(+)CD38(-) cells' self-renewal and multiple lineage potential, that were defined as "stemness" miRNAs. 12 of the 84 genes (14.29%) were common to 33 hematopoietic-expressed miRNAs expressed by CD34(+) cells from both peripheral blood and bone marrow in Georgantas's study, which included 10 upregulated miRNAs (hsa-miR-23b, -26b, -92, -107, -130a, -181a, -197, -213, -222, -223) and 2 downregulated ones (hsa-miR-16a, -155). Some "stemness" miRNAs undergo CD34 antigen-like expression pattern during development and commmitted differeniation of hematopoietic stem cell/progenitors. Hematopoiesis-associated miRNA clusters and putative target genes could be found with informatics technique. It is concluded that the hematopoietic "stemness" miRNAs play important roles in normal hematopoiesis: miRNA expression profiles of hematopoietic stem cell/progenitors --> their gene expression profiles --> their self-renewal and lineage-commmitted differeniation.

Objective: To study the clinical and pathologic characteristics of poorly differentiated cutaneous angiosarcoma of scalp., Methods: Eight cases of poorly differentiated cutaneous angiosarcoma of scalp were enrolled into this study. The clinical manifestations and histopathologic features were analyzed. Immunohistochemical study for CD31, CD34, factor VIII-related antigen, vimentin, AE1/AE3, CAM5. 2, epithelial membrane antigen and carcinoembryonic antigen was performed., Results: The mean age of the patients was 69 years. The male-to-female ratio was 5 : 3. The tumor manifested clinically as bruise-like lesion in early phase, indurated erythematous plaque accompanied by nodules, ulcerations and bleeding in advanced phase. Histologically, the tumor was composed of solid sheets of undifferentiated spindle cells which were not easily recognizable as vascular in origin. Nuclear atypia was always present. The tumor cells in all of the 8 cases strongly expressed CD31, factor VIII-related antigen and vimentin. Weak expression of CD34, AE1/AE3 and CAMS. 2 was noted in 2, 4 and 4 cases, respectively. The staining for epithelial membrane antigen, carcinoembryonic antigen and S-100 was negative. Conclusions Angiosarcoma needs to be excluded by histologic examination whenever bruise-like and erythematous lesions occurring on scalp skin of elderly patients. The endothelial origin of the tumor cells can be confirmed with immunostaining for CD31, CD34 and factor VIII-related antigen.

Objective: To study the cytotoxic effect of allogenetic natural killer (NK) cells in vitro on human CD34+ acute myelogenous leukemia cells., Methods: CD34 expression on acute myelogenous leukemia KG1a cells was detected by flow cytometry. KG1a cells were co-cultured at different effector-to-target (E:T) ratios with NK cells isolated from 5 healthy individuals using magnetic cell sorting. Lactate dehydrogenase (LDH) release assay was employed to examine the cytolysis of KG1a cells in the co-culture, and the inhibition rate of the KG1a cell colony formation in methylcellulose was determined with K562 cells sensitive to NK cells as the control., Results: A expression rate as much as (98.0-/+1.1)% was detected for CD34 antigen on KG1a cells, and the isolated NK cells (CD3(-)CD16+CD56+ cells) had a purity of (93.2-/+3.7)% after magnetic cell sorting. Allogenetic NK cells exhibited obvious cytotoxicity and colony inhibition in vitro against KG1a cells at different E:T ratios, and the effects were significantly enhanced as the E:T ratios increased (P<0.05). At the same E:T ratio, the cytotoxicity and colony inhibition rate of allogenetic NK cells against KG1a cells was lower than those against K562 cells (P<0.05)., Conclusion: Allogenetic NK cells exhibit obvious cytotoxicity and colony formation against CD34+ acute myelogenous leukemia cells.

Objective: To investigate vasculogenic potential of endothelial progenitor cells (EPCs) derived from human umbilical cord blood and their contribution to the neovascularization of malignant glioma in vivo., Methods: EPCs were isolated from human umbilical cord blood by density gradient centrifugation. After 7-10 days of culture, EPCs were investigated for CD34 and VEGFR-2 expression by direct immunofluoresent staining. The proliferative activity, migratory capability and forming capillary-like tubules were also monitored after stimulation with VEGF(50 mg/L) in vitro. Moreover, EPCs were administered into tumor-bearing mice, and the tumor and mouse organs were examined under confocal laser scanning microscope to visualize the distribution and localization of transplanted EPCs. In order to quantity the incorporation of EPCs into tumor vessels, cryosections of the tumor tissue were double-labelled with antihuman CD31 and anti-mouse CD31., Results: After 7 to 10 days of culture, EPCs assumed cobblestone-like monolayer growth pattern with nearly complete confluence, and expressed CD34 and VEGFR-2. Significant proliferative activity, increased migratory capability and forming capillary-like tubules were observed when stimulated with VEGF. The transplanted EPCs in vivo specifically homed to solid tumor tissue and incorporated into the tumor's endothelium. Quantitative analysis revealed that human EPCs contributed significantly to tumor neovascularization by incorporation into tumor vasculature (18.68 +/- 1.32)% of the total vessels., Conclusion: EPCs possess the potential to form neovascular network in tumor and play a role in the phenotypical heterogeneity of tumor microvascular architecture.

Objective: To identify and select smooth muscle progenitor cells from mouse bone marrow mesenchyme stem cell population and to characterize smooth muscle progenitor cells in peripheral blood., Methods: Recombinant expression vector with the promoter of sm22alpha was constructed to have an enhancement type green fluorescent protein expression plasmid (EGFP-1). The construct was transfected into mouse bone marrow mesenchyme stem cells using Lipofectamine 2000. Morphological assessment was performed and the expressions of myocardin at protein and mRNA levels by fluorescence microscope and RT-PCR were evaluated at 3, 5, 7, and 10 d targeting CD34 positive bone mesenchyme stem cells., Results: The transfection efficiency of the positive control group was 70% +/- 1.5% (P > 0.05). Expected green fluorescent proteins expressed at 3rd day. The numbers of green fluorescent cells in experimental groups increased with the time and reached the peak at the 7th day, and declined thereafter. The shapes of the green fluorescent cells were also different from each others. The positive ratios of green fluorescent cells at different time points: 3 d: 7% +/- 0.13%, 5 d: 10% +/- 0.32%, 7 d: 20% +/- 0.26%, 10 d: 12% +/- 0.18%, P < 0.05. Myocardin mRNA expression roughly correlated with green fluorescent expressions. CD34 was expressed on the 5th day in transfected bone mesenchyme stem cells. The CD34 positive ratio was 5.2% +/- 0.21% (P > 0.05)., Conclusions: There are smooth muscle progenitor cells among mouse bone marrow mesenchyme stem cell population. Smooth muscle progenitor cells can be selected using a Psm22alpha-EGFP-1 recombinant expression approach.

To explore the relationship between the expression of CD133 and pathogenesis of leukemia and MDS, immunocytochemistry method was used to examine the expression of CD133 in bone marrow cells of patients with leukemia and MDS. The results showed that the positive rate of CD133 in 41 acute leukemia patients was 51.2%. The expression of CD133 in AML patients (16/29, 55.2%) was significantly higher than that in control group (2/15, 13.3%). There was no significant difference in CD133 expression between CML and control group. The positive rate of CD133 in 9 patients with MDS was 55.56% (5/9). There was no significant difference between MDS and normal control. The expression of CD133 in all leukemia cells with CD34(+) was higher than that in leukemia cells with CD34(-), and there was significant difference in expression of CD133 between them (P < 0.05). The expression of CD133 had no relationship with the clinical prognostic factors such as sex, age, the percentage of leukemic cells in peripheral blood and in bone marrow, WBC counts, hemoglobin concentration, platelet counts and LDH level. It is concluded that the expression of CD133 in bone marrow cells of patients with AML is higher than that in control group. The expression of CD133 is significantly correlated with the expression of CD34. The high expression of CD133 may be an adverse prognostic factor in acute leukemia.

To construct a scFv antibody mini-library by phage display technique from the spleen cells of BALB/c mice immunized with extracellular domain of N-cadherin (N-cad), CD34 and AC133, the extracellular domain genes of N-cad, CD34 and AC133 were cloned into a phagemid pSEX81 respectively, and were then displayed on the phagemid in the form of fusion protein with p III protein. After being effectively immunized with the fusion protein, the spleen of the mice were harvested, and total RNA were extracted from the spleen. The cDNA of VH and VL genes were amplified by RT-PCR, and a scFv-phage display antibody library was constructed with the amplified V-genes. The content, multiplicity and expressing potential of the library were examined. As a result, we had produced a scFv library containing 1.4 x 10(6) individual clones which showed different patterns after being digested with restriction endoneuclease Mua I. The surface display expression of the library was also verified. It indicated that the capacity and diversity of the library was sufficient for screening specific scFv antibody against N-cad, CD34 and AC133. The library will be useful for isolating corresponding specific scFv antibodies.

Aim: To investigate the effects of both Th1/Th2 imbalance and its re-attainment on in vitro expansion and hematopoiesis of CD34+ cells from a severe aplastic anemia (sAA) patient., Methods: A preliminary-diagnosed sAA patient was studied. (1) Bone marrow mononuclear cells (BMMNC) were isolated and CD34+ and CD4+ cells were enriched by magnetic beads respectively. (2) The Th1/Th2 cell ratio within CD4+ subset was detected by flow cytometry (FCM). (3) Enriched-CD34+ cells were expanded and re-enriched for enough cells to be divided into four groups: control, Th cell treatment, Th cell and IFN-gamma treatment, and Th cell and IL-4 treatment, respectively. (4) After expansion for 10 d, colony-forming unit assay was performed on cells in each group. (5) Patient's Th1/Th2 ratio was followed up by FCM after immunotherapy., Results: (1) Symptom remission was achieved after therapy for 5 months. (2) Th1/Th2 cell ratio of the patient before and after remission was 22.47 and 12.27, respectively, while that of healthy controls was 8.98+/-4.45. (3) The CD34+ cell expansion rates as well as CFU numbers, from high to low, were ranked as control, Th cell and IL-4 contained group, Th cell contained, and Th cell and IFN-gamma contained group., Conclusion: Predominant Th1 cells seem to directly inhibit the self-renewal, proliferation and lineage differentiation of CD34+ cells in vitro, which can be counteracted by IL-4, probably mediated by switching Th1/Th2 balance.

Objective: To compare the leukemia-associated immunophenotypes (LAIP) in patients with B lineage acute lymphoblastic leukemia (B-ALL) at diagnosis and relapse, and investigate its implications for minimal residual disease (MRD) detection., Methods: The immunophenotype of leukemia cells from 410 newly diagnosed and 6 relapsed patients with B-ALL were detected by four to six antibody combination, mainly CD34/CD10/CD45/CD19 of 4-color CD45/SSC gating flow cytometry (FCM)., Results: The proportion of CD45 under-expressed or negative in relapsed patients was much higher than that in newly diagnosed patients, being 69.2% and 37.8% respectively. Immunophenotypic changes occurred in 9 relapsed patients (including 8 hematological relapse and 1 central nerves system relapse) when analyzed by paired samples analysis at diagnosis vs at relapse: 4 cases showed CD45 down-modulation and 2 up-modulation; 4 CD34 down-modulation and 2 CD10 up-modulation, while the expression of CD19 remained no change. MRD was observed in all 7 cases of hematological relapse 2 - 4 months before relapse, and the immunophenotype of MRD cells was the same as that in relapse., Conclusion: A high frequency of immunophenotypic changes occurred at relapse and even in MRD before relapse, however the accuracy of MRD monitoring seemed not affected by the FCM strategy used in this investigation.

Objective: To evaluate the clinical significance for minimal residual disease (MRD) detection by 4 color flow cytometry in B lineage acute lymphoblastic leukemia (B-ALL)., Methods: MRD was analyzed and followed up by using two panels of 4 color antibodies, mainly CD34/CD10/CD45/CD19, in 671 consecutive bone marrow specimens and 1 cerebrospinal fluid from 98 B-ALL patients. In 26 cases of them the immunophenotyping informations at diagnosis were not available., Results: Of 671 bone marrow samples, 579 were MRD negative with leukemic cells below 0.0001 and 93 were MRD positive with leukemic cells over 0.0001. Of 93 MRD positive samples, leukemic cells below 0.05 were found in 64 bone marrow samples, meanwhile in the other 29 samples leukemic cells were over 0.05. Twenty patients relapsed, 19 were bone marrow relapse and one center nerves system. Fifteen of them were found MRD positive 7 - 17 weeks before relapse including 6 patients having no immunophenotyping data at diagnosis. The percentages of leukemia cells in these 15 patients were all over 0.0001. Two relapsed patients were MRD negative in 3 and 9 months before relapse, respectively. Two relapsed after MRD monitoring stopped. If MRD level was > 0.0001 at the end of induction chemotherapy and 12 weeks of treatment, the rate of relapse was 50% (6/12), while, it was 7.5% (3/40) in MRD negative patients (P = 0.000)., Conclusion: Relapses can be predicted by MRD monitoring, if MRD was positive in the early phase of treatment, the risk of relapse was higher. Based on the characteristics of B cells ontogeny, MRD detection can be done independently of immunophenotypic information at diagnosis.

To investigate the effects of stromal cell-derived factor 1 (SDF-1) and platelet factor 4 (PF4) on the homing-related function of expanded ex vivo umbilical cord blood CD34(+) cells, purified cord blood CD34(+) cells were cultured in serum-free medium containing a HGF combination of FL + SCF + TPO (FST) with either 100 ng/ml SDF-1 alone, 100 ng/ml PF4 alone, or both of these 2 cytokines. The expansion rate of CD34(+) cells, colony formation, homing-related functions including expression of homing-related adhesion molecules of expanded CD34(+) cell, adhesion activity and chemotactic function of the re-selected expanded CD34(+) cells were evaluated at different time points. The results showed that expansion rate of CD34(+) cells and expansion multiple of CFU in SDF-1 groups were higher than those in control. The expression of CD49e on the expanded CD34(+) cells was remarkable up-regulated, in contrast, expression of CXCR-4 on the expanded CD34(+) cells was remarkable down-regulated in SDF-1 groups. The expression of CD49e, CD54 and CXCR-4 on the expanded CD34(+) cells were remarkably up-regulated in the PF4 groups. In all the SDF-1 group, PF4 group and SDF-1 plus PF4 group, the ability of expanded CD34(+) cells adhering to fibronectin layer were higher than those in the control on day 10. Spontaneous migration rate of expanded CD34(+) cells in SDF-1 groups were higher than those in control, while SDF-1-induced migration rate were lower than those in control on day 10. SDF-1-induced migration rate in PF4 groups were higher than those in control on day 10. Spontaneous and SDF-1-induced migration rate of expanded CD34(+) cells in the SDF-1 plus PF4 groups were higher than those in control on day 10. It is concluded that, SDF-1 and PF4 can up-regulate expression of adhesion molecules on expanded CD34(+) cells, and retain the adherent and migration ability of expanded CD34(+) cells, which is helpful for the homing of expanded CD34(+) cells. In short, SDF-1 and PF4 are helpful for the homing-related function of the expanded UCB HSPC.

Objective: To explore the role of stem cell mobilization on regeneration of partially grafted livers., Methods: Rats models with cross-sex 50% PLTx (partial liver transplantation) were established. The rats were divided into three groups: PLTx, WLTx (whole liver transplantation) and sham operation groups. Bone marrow and liver samples were collected on days 1, 3, 5, 7 postoperatively (each n = 6). The quantitative variations of the cells with stem cell markers in the bone marrow, including beta2m-/Thy-1.1+, CD45+/CD34+, Flt2/3+ and c-kit+ markers, were detected using flow cytometry. Sry gene positive cells in donor livers were detected by fluorescent in situ hybridization (FISH), and the expressions of CD34, c-kit and Thy-1.1 were detected by immunohistochemistry technique., Results: Compared with the WLTx and sham operation groups, beta2m-/Thy-1.1+, CD45+/CD34+ cells in bone marrows in the PLTx group increased on the first postoperative day and decreased on the following days. The CD34, c-kit and Thy-1.1 positive cells detected in portal tract areas peaked during the 3-5 postoperative days. CD34+/CD45+ positive cells could be detected. The expressions of CD34, c-kit and Thy-1.1 positive cells were rare in the WLTx and sham operation groups. Sry+ cells could be detected in portal tract areas and few Sry+/CD34+ and Sry+/Thy-1.1+cells were detected., Conclusion: In the PLTx group, the stem cells in the bone marrow were mobilized and stem cells in the liver were activated.

To study the effect of interleukin-15 (IL-15) on the proliferation, differentiation and apoptosis of MDS CD34(+) cells, CD34(+) cells of high enrichment were separated by MACS system, and cultured in liquid media with different concentration of IL-15 in treated group and without IL-15 in the control group. Apoptosis of hematopoietic precursors was assayed by propidium iodine staining and cell by FCM, and the other MDS CD34(+) cells were stained by cytochemical staining after culture. The results showed that after culture with IL-15 the proliferation and differentiation of MDS CD34(+) cells were obviously promoted. It was found the every lineage of mature cells developed, the expressions of cell surface antigens CD71, CD33 and CD19 all increased in the MDS CD34(+) cell treated with IL-15. It is suggested that IL-15 stimulates the proliferation and differentiation of MDS CD34(+) cells, and partly shows anti-apoptosis effects which may be applicable to the therapy MDS.

Objective: To evaluate the method for expanding Valpha24 natural killer T (NKT) cells with alpha-galactosylceramide (alpha-GalCer) ex vivo., Methods: Mononuclear cells (MNCs) isolated from adult peripheral blood or umbilical cord blood (UCB) were divided into three groups. In Group A1 (n = 5), CD34+ progenitorderived dendritic cells were differentiated in a cytokine-supplemented culture system from cord blood and acted as antigen presenting cells (APC) to induce the expansion of cord blood Valpha24 NKT cells in presence of alpha-GalCer; in Group A2 (n = 5), adult peripheral monocyte-derived dendritic cells (Mo-DC) were used as APC to induce the expansion of adult peripheral NKT cells in presence of alpha-GalCer; whereas in Group B (n = 16), alpha-GalCer was added into adult peripheral MNCs culture system without additional DCs. Cytokine-produce were measured by ELISA, and NKT cells' proliferation ability, cytotoxicity, and suppressive effect on mixed lymphocyte reaction (MLR) were examined by MTT assays., Results: Valpha24 NKT cells in Group A1, A2, and B were expanded up to 128 (95-207), 250.5 (179.6-790.6), and 326 (101-2 136) -fold by day 12, respectively. Adult NKT cells expanded in Group B were markedly better than those in Group A1 (P = 0.038). When stimulating by PMA, the NKT cells had a 3-day stimulate index of 1.80 +/- 0.41; and the secretion ratio of IL-4 to IFN-gamma of UCB or adult peripheral blood NKT cells were 0.30 +/- 0.13 and 0.28 +/- 0.18; and the ex vivo antitumor effect of expanded NKT cells were found in cell line HL60, KG1a, and Raji except for K562; and the suppressive effect of expanded NKT cells or the culture supernatant on MLR were confirmed., Conclusions: Alpha-GalCer can facilitate the rapid shorttime expansion of Valpha24 NKT cells in presence of IL-2 and IL-15. These expanded NKT cells, kill tumor cell lines, and inhibit can massively excret IL-4 and IFN-gamma allogeneic T-cell response.

Objective: To cultivate hematopoietic stem/progenitor cells (CD34(+)CD38(-)) isolated from umbilical cord blood (UCB) long for the observation of cell growth and expansion in vitro, surface marker expression, and chromosomal complements., Methods: By flow cytometry CD34-FITC and CD38-PE labeled CD34(+) and CD38(-) stem/progenitor cells were isolated from UCB. The cells were cultivated in vitro for 6 months in a stem cell culture system with addition of six kinds of cell growth factors (IL-3, IL-6, GM-CSF, Epo, SCF, IGF-1). One month after cultivation, cultured cells were investigated for surface marker expression by flow cytometry and karyotype by G banding method., Results: After 7-12 days cultivation, the CD34(+)CD38(-) stem/progenitor cells began proliferation. The proliferation rate and the peak proliferation duration were greater in 1 cell/well cultivation conditions than in 10 cells/well. The cells remained CD34(+)CD38(-) and their karyotypic characteristics remained unchanged., Conclusion: CD34(+)CD38(-) stem/progenitor cells from UCB may provide a larger than original amount of stem/progenitor cells for transplantation after long-term cultivation in vitro.

Objective: To compare the expression profiles of a set of homing-related molecules (HRM) repertoire expressed on hematopoietic stem/progenitor cells (HS/PC) from different sources., Method: The expression levels of HRM on HS/PC from umbilical cord blood (UCB), mobilized peripheral blood (mPB) and bone marrow (BM) were assessed using a highly sensitive 4-color flow cytometric analysis., Results: UCB-derived CD34(bright) cells, as well as mPB- and BM-derived CD34(bright) cells strongly expressed CD44, CD11a, CD18, CD62L, CD31 and CD49d. On the other hand, significantly lower expressions of CD49e, CD49f, CXCR-4 and CD54 on UCB-derived CD34(bright) and CD34(bright)CD38(-) cells, compared with those on mPB- and BM-derived CD34(bright) and CD34(bright)CD38(-) cells, were observed. None of UCB-, mPB- and BM-derived CD34(bright) cells expressed other chemokine receptors, including CCR-1, CCR-2, CCR-3, CCR-5, CXCR-1, CXCR-2, CXCR-3 and CXCR-5. Another striking finding was that only mPB-derived CD34(bright) cells expressed significant levels of both the matrix metalloproteinases MMP-2 \[(11.4 +/- 4.9)%\] and MMP-9 \[(27.6 +/- 7.8)%\]., Conclusion: HS/PC from UCB have some defects of expression of HRM repertoire, which might partly explain the cause(s) of delayed hematopoietic reconstitution after UCB transplant.

In order to find a method suitable for purifying large amount of CD34(+) cells, from 5 cases who accepted autologous peripheral blood stem cell transplantation, CD34(+) cells were collected and enriched by using Isolex 300i (Nexell). Phenotypes were detected by flow cytometry and the biological viability were assayed by the colony-forming experiments and cell expansion experiment in vitro. The results showed that the number of mononuclear cells first collected was about (3.5 - 6.0) x 10(10) and (0.55 - 1.2)% of cells were CD34 positive. The number of positive production was about (2.0 - 3.0) x 10(8); the CD34(+) cells purity was (75 - 85)% and the yield was (40 - 65)%. The CD34(+) cells of positive production could expand up to 2 - 3 times when cultured with SCF + IL3 + FL + TPO + EPO in vitro. The results of colony-forming experiments demonstrated that the CD34(+) cells collected has enough colony-forming ability. All results showed the enriched CD34(+) cells with biological viability. In conclusion, the CD34(+) immunomagnetic isolation apparatus Isolex300i is suitable to clinical application for a large amount of CD34(+) cell enrichment.

Objective: To investigate the roles of different cells in the pulmonary lesions in the severe acute respiratory syndrome (SARS) patients., Methods: The monoclonal antibodies of CD8, CD20, CD34, LCA, CD56, CD68, and AE1/AE3 are used to demonstrate the different cells in the lung specimens of SARS patients in order to study the patterns of cell responses in this new disease. Meanwhile the HE stained slides were also carefully studied to compare with the results of immunohistochemical staining., Results: The number of capillaries increased and the capillaries clearly outlined the contour of alveolar wall from beginning to early stage of organization, the number of lymphocytes decreased sharply while the number of macrophage remarkably increased, together with proliferation of type II pneumocytes. The numbers of blood vessels decreased in the fibrotic and consolidated lung tissue, and the vessel cavities enlarged, losing the normal contour of alveolar septa., Conclusions: The lesions in the lung from SARS patients are consisted of the tissue reaction to the inflammatory injury, including extensive exudation, capillary proliferation, fibrosis, and obvious infiltration of macrophages which may play a key role in the pathogenesis of pulmonary lesions of SARS.

Objective: To study the effect of ex vivo expansion on the adhesion activities and chemotactic function of umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPCs)., Methods: CD34(+) cells isolated from fresh UCB samples were cultured in serum-free and stroma-free culture system. After 7, 10 and 14 days' culture, CD34(+) cells were re-selected from the expanded products. Stromal cell- derived factor-1 (SDF-1) 100 ng/ml was added into the experimental CD34(+) cells and the absorbance at 570 nm of all groups was examined. 20 micro g/ml fibronectin (Fn) was added and the spontaneous adhesion between CD34(+) and FN was detected by MTT method. The homing-related functions including expression of homing-related adhesion molecules (CAM), adhesion activity and chemotactic function of the re-selected CD34(+) cells were evaluated and compared with those of the initial fresh CD34(+) cells., Results: (1) The expression of CD49d, CD44, CD11a and CD49e on expanded CD34(+) cells increased or sustained the same levels as those of the fresh isolated UCB CD34(+) cells, while the expression of CD62L, CD54 and CD31 on expanded CD34(+) cells declined during the culture. (2) The spontaneous adhesion between CD34(+) and FN and SDF-1-induced adhesion continuously increased in the course of the first 10-day culture. The spontaneous adhesion rate and SDF-1-induced adhesion rate on day 0, day 7 and day 10 were 28% and 63%, 60% and 70%, 63% and 90% respectively. (3) The migration efficiency of re-selected CD34(+) cells on day 7 was almost the same compared to that of fresh CD34(+) cells., Conclusion: The expanded HSPCs sustain most of the homing-related characteristics and activities during one-week culture while extended culture may partly impair their intrinsic homing potential.