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This study was aimed to investigate whether aspirin has effect on function of late endothelial progenitor cells (EPC). Cord blood CD34(+) cells were purified using the ficoll density gradient centrifugation and human CD34 positive selection kit, then the cells were inoculated on fibronectin-coated culture plate. After culture for 2 weeks, adherent cells were identified as EPC by flow cytometry, immunofluorescence, RT-PCR, uptake of Dil-Ac-LDL and matrigel tube formation assay. EPC were treated with different concentrations of aspirin (0.1, 1, 10, 100, 1 000, 10 000 µmol/L) for 24 h, then the proliferation, adhesion and migration ability of these cells were analyzed by CCK-8 assay and transwell methods. The results indicated that the low concentrations of aspirin (0.1 and 1 000 µmol/L) promoted late EPC adhesive and migratory capacity, but no obvious effect on proliferation of late EPC were observed. On the other hand, the high concentrations of aspirin (10 000 µmol/L) inhibited proliferation and migratory capacity of EPC, but had no obvious effect on adhesive ability of EPC. It is concluded that low concentration of aspirin promotes migration and adhesion of late EPC, while the high concentration of aspirin decreases EPC proliferation and migratory capacity of EPC.

Interdigitating dendritic cell sarcoma (IDCS) is an extremely rare tumor derived from interdigitating dendritic cells. We report the first case of a 64-year-old Chinese woman who was diagnosed with simultaneous IDCS and acute myelomonocytic leukemia (AML-M4). The patient had undergone chemotherapy for breast cancer 6 years previously. Based on the laboratory results, both the IDCS and the AML-M4 in this patient were determined to be of myelogenous origination. Furthermore, a review of 62 IDCS cases (Medline database, key word: IDCS) reported to date revealed that as many as 17 % of the patients had malignant disease and received radiotherapy and/or chemotherapy prior to developing IDCS, and that this group of patients showed worse prognosis compared with counterparts. The patient in the present report showed poor response to four cycles of sequential chemotherapy, and died 6 months after the initial diagnosis.

Objective: To investigate the susceptibility of von Willebrand factor (VWF) type 2A mutant A1500E to proteolysis by metalloprotease ADAMTS13 and to provide the direct supports for the pathogenesis of VWF mutation A1500E responsible for von Willebrand disease (VWD) type 2A., Methods: Recombinant wild-type VWF (WT-VWF) and A1500E mutant VWF transiently expressed on transfected HeLa cell lines. Expression media were collected and concentrated, then cleaved directly by recombinant ADAMTS13 (rADAMTS13). Compared with WT-VWF, the susceptibility of A1500E mutant VWF to proteolysis by ADAMTS13 was analyzed using SDS-agarose gel VWF multimers analysis., Results: In vitro the expression of VWF:Ag in the supernatants of WT-VWF and A1500E mutant VWF were 1.10 U/ml and 0.78 U/ml, respectively, while VWF:Ag in cells lysates of A1500E mutant VWF was 90.6% of that of WT-VWF. The SDS-agarose gel VWF multimers analysis showed that there were no differences between WT-VWF and A1500E mutant VWF. The A1500E mutant VWF could be efficiently cleaved by ADAMTS13 under static condition without denaturants such as urea and guanidine HCl. VWF multimeric analysis showed that high and intermediate molecular weight multimers dramatically decreased while low molecular weight multimers obviously increased. Conversely, WT-VWF could not be cleaved by ADAMTS13 under the same condition., Conclusion: The A1500E mutation resulted in VWF more susceptible to ADAMTS13-dependent proteolysis, which belonged to VWD type 2A group 2 mutation.

Objective: To construct the expression vectors of vWF73 and vWF114 fragments of von Willebrand factor (vWF) A2 domain, and to express glutathione S-transferase (GST) fusion proteins in E. coli, and to explore their values in measuring ADAMTS13 activity as substrates., Methods: The DNA fragments encoding vWF73 and vWF114 were generated using PCR and separately cloned into pGEX-6P-1, a Schistosoma japonicum GST fusion expression vector. The expression of GST-vWF73-H and GST-vWF114-H was induced in liquid culture, followed by purification with Ni-NTA agarose column. The cleavage of two GST fusion proteins by recombinant ADAMTS13 (rADAMTS13) or plasma from normal individuals and thrombotic thrombocytopenic purpura (TTP) patients were identified by Western blot. Based on an enzyme-linked immunosorbent assay (ELISA) with anti-GST and anti-His monoclonal antibodies, GST-vWF73-H and GST-vWF114-H were used to measure plasma ADAMTS13 activity as substrates., Results: Two small molecular substrates of ADAMTS13, GST-vWF73-H and GST-vWF114-H, are expressed and purified, which could be specifically cleaved by rADAMTS13 or plasma from healthy individuals, but not by plasma from congenital or idiopathic TTP patients. An ELISA assay was established to detect plasma ADAMTS13 activity using GST-vWF73-H and GST-vWF114-H as substrates., Conclusions: Two GST fusion proteins in vWF A2 domain, vWF73 and vWF114, were expressed effectively using a prokaryotic expression system and could be used to detect ADAMTS13 activity as substrates.

Objective: To establish NB4/VEGF-C cells xenograft in nude mice model, and explore the effect of VEGF-C on hematological malignancies, Methods: NB4/VEGF-C or NB4/pcDNA3.1 cell lines were established by transfecting the recombinant pcDNA3.1-VEGF-C plasmid and the vacant pcDNA3.1 vector into NB4 cells. The recombinant VEGF-C was identified by RT-PCR and Western blotting. Eighteen male BALB/c nude mice aged 4 - 5 weeks were equally divided into two groups. Mice irradiated by 4 Gy ⁶⁰Co were subcutaneously injected with 1 × 10⁷NB4/VEGF-C or NB4/pcDNA3.1 cells into one side of axilla. The volumes of xenograft tumor was evaluated according to L × t² × 0.52. Microvessel density (MVD) on the xenograft tumor section was detected by IHC with VWF antibody., Results: NB4 cell xenograft tumors were developed in all mice of both the two groups. The growth of NB4/VEGF-C cells in nude mice was faster than in controls. There were statistically significant differences in the volume and weight of xenograft tumor between NB4/VEGF-C and NB4/pcDNA3.1 cell groups \[(631.44 ± 114.42) mm³ vs (491.22 ± 70.05) mm³\] (P = 0.006) and \[(321.78 ± 27.84) mg vs (288.57 ± 40.12) mg\] (P = 0.031), respectively. MVD in xenograft tumor of NB4/VEGF-C cells \[(50.8 ± 11.7)/mm²\] was higher than that in controls \[(18.9 ± 7.0)/mm²\] (P = 0.021). The Bcl-2 protein level in NB4/VEGF-C cells xenografts was higher than that in controls., Conclusion: VEGF-C could promote proliferation of NB4 cells by inducing angiogenesis and inhibit cells apoptosis by upregulating antiapoptotic Bcl-2 protein expression in NB4 cells xenograft tumor.

Objective: To study the influence of C-terminal domain of ADAMTS13 on its cleaving activity., Methods: The full-length wild-type (WT) and C-terminal domain truncated type (TT, TSP8 + CUB domains were deleted) of human ADAMTS13 recombinant protein were transfected into and permanent expressed on Hela cells. Western blot and R-CBA were used to directly detect the activities of the two recombinant proteins under the static and stressed condition respectively. ELISA was used to compare the binding abilities of the two proteins by coating with vWF., Results: The recombinant proteins were identified by Western blot with anti-his-tag or anti-ADAMTS13 antibodies. With pretreatment of 1.5 M urea, the enzyme activity of TT was significantly higher than that of WT, and so did in binding ability with vWF While, only WT could cleave vWF under high stress., Conclusion: The distal carboxyl-terminal TSP8 together with CUB domains of ADAMTS13 may affect the enzyme activity by regulating the binding of ADAMTS13 to vWF in different conditions, and they are very important for the enzyme activity under high stress force condition.

Background: A severe deficiency of ADAMTS13 activity contributes to the pathogenesis of thrombotic thrombocytopenic purpura (TTP). Measuring the activity of ADAMTS13 is helpful for the diagnosis of TTP and the prognostic monitor in TTP patients. Most available assays are cumbersome and costly, so not easily adapted to routine laboratories. ADAMTS13 cleaves von Willebrand factor (VWF) within the domain A2, located between domains A1 and A3. Therefore, specific assays for ADAMTS13 activity could be based on the different structures of VWF before and after the cleavage. Using this hypothesis we try to establish a new and simple method to determine ADAMTS13 activity., Methods: First, plasma samples were exposed in denaturing condition to allow cleavage of VWF by ADAMTS13. Then, the ADAMTS13 activity was measured with two novel monoclonal antibodies, SZ-129 and SZ-125, which specifically recognize the VWF A1 and A3 domains by using a two-site sandwich ELISA. Compared with a residual-collagen binding assay (R-CBA), plasma ADAMTS13 activities in 161 samples were assessed, and the inhibitory activities of ADAMTS13 autoantibody in 24 TTP patients were determined. The relationship of these two assays was analyzed by linear correlation, and the sensitivity and specificity of the new assay was also evaluated., Results: Plasma ADAMTS13 activities in normal people and TTP, acute myocardial infarction (AMI), and idiopathic thrombocytopenic purpura (ITP) patients determined by the new assay were (89.75 +/- 7.93)%, (17.63 +/- 18.71)%, (68.55 +/- 18.08)%, (85.83 +/- 9.84)%, respectively. Results were consistent with those of R-CBA, the squared correlation factor was 0.9183 of the two assays. The new assay can easily discriminate a TTP plasma sample from a non-TTP plasma sample (P < 0.01), and the coefficient of variation for the new assay was 6.17%. In 23 idiopathic TTP patients, the inhibitor activity of ADAMTS13 autoantibody ranged from 12% to 100%, while no inhibitory activity was detected in one hereditary TTP patient., Conclusion: This new and simple assay for ADAMTS13 activity could be used routinely in the clinic to determine the activity of ADAMTS13.

Objective: To analyze the phenotype and genotype of a family with inherited dysfibrinogenemia., Methods: Assays of coagulation, including activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT), were carried out with Stago Compact in the proband and his family members. The activity and antigen of fibrinogen in plasma were determined by Clauss and immunoturbidimetry, respectively. Fibrinogen and its constituent were analyzed by Western blot with nonreducing 4%-20% SDS-polyacrylamide gel electrophoresis (PAGE). All exons and exon-intron boundaries of fibringen genes FGA, FGB and FGG were analyzed by PCR and then direct sequencing., Results: The proband had normal APTT and PT, but prolonged TT. The activity of fibrinogen in plasma was decreased while its antigen level was normal. These abnormalities were also found in his mother and a sister. Genetic analysis revealed heterozygous G1233A in the exon 2 of FGA originating from his mother, which resulted in Arg16His missense mutation., Conclusion: Inherited dysfibrinogenemia was caused by Arg16His mutation in exon 2 of FGA, and this is the first case reported in a Chinese family.

Objective: To investigate the effects of Annexin II (AnxA2) gene silencing by siRNA on proliferation and invasive potential of lymphoma cell line Jurkat cells., Methods: A synthesized siRNA duplex targeting to AnxA2 was transfected into Jurkat cells. Transfection efficiency was analyzed by real-time PCR and flow cytometry. MTT assay for cell proliferation and transwell plates for invasive potential were performed., Results: Compared with the negative controls, the cell proliferation inhibitory rate of the AnxA2 siRNA transfected Jurkat cells was significantly increased at 24 h, 48 h and 72 h [(17.4 +/- 2.3)%, (22.4 +/- 3.8)%, (37.6 +/- 1.5)% vs (-1.3 +/- 5.1)%, (-5.5 +/- 4.4)%, (-10.8 +/- 5.5)%, respectively, P<0.05]. The cell invasive potential of the transfected Jurkat cells was inhibited remarkably at 48 h (11.3 +/- 4.2 vs 54.3 +/- 8.7, P<0.01)., Conclusion: AnxA2 gene silenced by siRNA can inhibit the proliferation and the invasive potential of Jurkat cells remarkably.

Objective: To develop a monoclonal antibody (mAb) directed to FVIII C2 domain and investigate its effect on FVIII activity., Methods: FVIII C2 protein was expressed in E. coli and purified. A murine antihuman FVIII C2 domain mAb SZ-132 was developed by standard hybridoma technology and characterized. In coagulation assays, different concentrations of SZ-132 were incubated with freshly collected pooled human plasma and the residual activity of FVIII and activated partial thromboplastin time (APTT) were determined. The effects of SZ-132 on rhFVIII binding to purified human vWF, phosphatidylserine (PS) and platelets were assessed by enzyme linked immunosorbent assays (ELISA)., Results: SZ-132 could inhibit FVIII procoagulant activity in a dose-dependent manner within the concentrations of 0-25 microg/ml and the FVIII activity was completely inhibited on above 25 microg/ml. It could also prevent rhFVIII from binding to vWF, PS and platelets., Conclusions: SZ-132 is a neutralizing mAb against FVIII C2 domain and can inhibit FVIII procoagulant activity by preventing FVIII from binding to vWF and PS.

Aim: To generate and screen the specific Fab phage antibody library against human Daudi cell strain in B-lymphoma and identify the positive clones., Methods: BALB/c mice were immunized with Daudi cells, and antisera were titrated by ELISA. Following the demonstration of sufficient antibody titer, total RNA was extracted from splenic lymphocytes of the immunized mice and RT-PCR was used to amplify kappa light chain and Fd fragments of heavy chain. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the kappa light chain and the Fd fragments were successively inserted into the phagemid vector pComb3H-SS and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Daudi cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. Following six rounds of biopanning with Daudi cells, the antigen binding activities of random clones were tested by ELISA to select the positive clones, which were further DNA sequenced, expressed in E.coli XL1-Blue and identified by Western blot., Results: The Fab phage antibody library with 3.13x10(7) size was constructed and four positive clones which specifically recognized Daudi cell strain were isolated. In amino acid sequences, the variable heavy domains (V(H)) were found to be 80%-02.394% and variable light domains (V(L)) 88%-95% homologous with respective murine germline genes in GenBank. Furthermore, soluble Fab antibodies of the positive clones were successfully expressed in E.coli XL1-Blue and the reactivity with the membrane proteins of Daudi cells was demonstrated by Western blot., Conclusion: Fab phage antibody library is successfully constructed and specific antibodies against membrane antigens in Daudi cells are obtained, which provides an experimental foundation for the further investigation of B-lymphoma immunotherapy.

This study was aimed to investigate the expressions of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) in patients with multiple myeloma (MM) and its clinical significance. Expression of VEGF was detected by enzyme linked immunosorbent assay (ELISA) and the level of COX-2 was detected by Western blot. The results showed that the serum VEGF level of multiple myeloma patients (365.34 +/- 65.63 pg/ml) was higher than that in the normal persons (122.52 +/- 39.29 pg/ml) (p < 0.05); the serum VEGF level of patients at advanced stage (395.07 +/- 54.90) pg/ml was higher than those at stable stage (300.33 +/- 44.22) pg/ml (p < 0.05). The serum Cox-2 positive rate in the patients (31%) was higher than that in normal persons (0%) (p < 0.01); the serum Cox-2 positive rate in the patients at advanced stage (50%) was higher than those at stable stage (21%) (p < 0.01). It is concluded that VEGF and COX-2 may play an important role in the pathogenesis and development of multiple myeloma, they can be used to evaluate the status of patients with MM.

Aim: To generation of human Fab fragment against GP GPIIb/IIIa from phage display library and to study its effect on platelet aggregation., Methods: An antibody phage display library was constructed from spleen cells from a donor with idiopathic thrombocytopenic purpura(ITP) whose anti-GPIIb/IIIa antibody was positive. High-affinity human mAbs was selected by panning against GPIIb/IIIa expressed on CHO123 cells. The binding specificity of phage antibody to GPIIb/IIIa was detected by ELISA and Western blot. And the effect of antibody on platelet aggregation was analyzed., Results: After three rounds of panning with CHO123, phage antibodies against GPIIb/IIIa were enriched specifically. 2 positive phage antibodies with high specific for GP IIb/IIIa were verified, and the purified antibody can inhibited ADP induced platelet aggregation., Conclusion: Human antibodies against GPIIb/IIIa are obtained from phage display library.

Aim: To record funny currents (If) of ventricular myocytes and to analysize hyperpolarization-activated cation channel(HCN) expression in the rats of different ages., Methods: Fresh ventricular myocytes were isolated from 3 days rats and adult rats.HCN expressions were measured by real-time quantitative polymerase chain reaction(real-time PCR). It was recorded through whole-cell patch clamp., Results: HCN1, HCN2, HCN3, HCN4 mRNA represented 0.23% +/- 0.01%, 83.58% +/- 0.04%, 0.79% +/- 0.01%, 15.44% +/- 0.01% of total HCN mRNA in the neonatal rats, respectively. If was recorded and the threshold for activation was -75 mV. In the adult rat, HCN1, HCN2, HCN3, HCN4 mRNA represented 0.72% +/- 0.02%, 91.58% +/- 0.08%, 0.27% +/- 0.02%, 7.12% +/- 0.02% of total HCN mRNA. The ratio of HCN2 to HCN4 was approximately (13.06 +/- 0.21):1. The threshold for activation of If was approximately -115 mV in the adult rats., Conclusion: With the development of rats, the value of If is smaller. The threshold for activation of If is more negative. The ratio of HCN2 to HCN4 is bigger.

Objective: To investigate the ADAMTS13 antigen levels and activity in patients with acute myocardial infarction (AMI) and acute ischemic stroke (AIS), and explore its significance in these diseases., Methods: ADAMTS13 activity levels were detected by a new developed Frests-vWF73 kit, ADAMTS13 antigen levels by ELISA kit, and vWF multimers by electrophoresis., Results: ADAMTS13 antigen in normal control, AMI and AIS was (878 +/- 198), (618 +/- 188) and (702 +/- 155) U/L, and ADAMTS13 activity was (81.7 +/- 13.9)%, (59.2 +/- 22.1 )% and (65.4 +/- 15.8)%, respectively, being significantly decreased in AMI and AIS patients., Conclusion: ADMATS13 might involve in arterial infarction diseases.

Objective: To prepare anti-von Willebrand factor A3 (vWF-A3) domain monoclonal antibodies(mAbs) which block vWF-A3 binding to collagen, and characterize their biochemical properties and functions., Methods: BALB/c mice were immunized with purified recombinant vWF-A3 protein (rvWF-A3). Murine anti-human vWF-A3 mAbs were developed by standard hybridoma technology and identified with ELISA. The recognition of the mAbs with rvWF -A3 and reduced human vWF was identified by Western-blot. The effect of mAbs on binding of purified human vWF to human placenta or calf skin collagen III was studied with collagen binding inhibition test., Results: A group of 30 murine anti-human vWF-A3 mAbs was obtained, from which 2 clones were identified as inhibitory ones and designated as SZ-123 and SZ-125. SZ-123 and SZ-125 could react specifically with human vWF and rvWF-A3 respectively, while neither of them reacted with rvWF-A1 and rvWF-A2. Western-blot showed that SZ-123 and SZ-125 could recognize a 27 x 10(3) band of rvWF-A3 and 2 reduced human vWF bands at 250 x 10(3) and 170 x 10(3). SZ-123 and SZ-125 not only inhibited the binding of purified human vWF (1.5 and 3.0 microg/ml) to human type III collagen and to calf skin collagen III in a dose dependent manner, but also inhibited the binding of plasma vWF from human, rhesus monkeys or Beagle dogs to the two collagens., Conclusion: SZ-123 and SZ-125 are neutralizing mAbs against vWF-A3 domain and may have therapeutic potential as an antithrombotic agent.

Background & Objective: Mer is a member of Axl receptor tyrosine kinase family; its ligand Gas6 can bind Mer, then stimulate the tyrosine kinase activity and downstream cell signal pathway of Mer, and take part in cell inflammation and apoptosis. There are more and more reports on Mer function, while few on its association with malignant diseases. This study was to detect the expression of Mer on T-cell leukemia cell line Jurkat, and investigate its anti-apoptosis function and the mechanism., Methods: Flow cytometry (FCM) was used to detect Mer expression on normal T cells and Jurkat cells. RNA interference (RNAi) was used to block the expression of Mer on Jurkat cells. The effect of Mer on the proliferation of Jurkat cells was assessed by MTT assay, and its effect on serum starvation-induced apoptosis was evaluated by FCM with Annexin V/PI double staining. The expression of apoptosis-associated genes Bcl-2 and Caspase-3 in Jurkat cells was detected by real-time polymerase chain reaction (PCR) after Mer blocking., Results: Mer was not expressed on normal T cells both from peripheral blood and bone marrow, but highly expressed on Jurkat cells with a positive rate of 51.1%. The inhibition rate of Mer expression on Jurkat cells by RNAi was 86.0%. After 48-hour serum starvation, the apoptosis rate was 15.3% in Mer-blocking Jurkat cells, and only 1.5% in control Jurkat cells. There was no significant difference in the proliferation rate of Jurkat cells between these 2 groups. After Mer blocking, Bcl-2 expression was decreased by 42.7% of control, Caspase-3 expression showed no significant change., Conclusion: Mer is highly expressed on Jurkat cells, and could inhibit cell apoptosis via Bcl-2 signaling pathway.

Objective: To analysis the effect of amiodarone on funny current (I(f)) and hyperpolarization-activated cation channel (HCN) gene expressions of the neonatal rat ventricular myocytes., Methods: Ventricular myocytes of 1 - 3 days-old rats were isolated and cultured. The cardiomyocytes were treated by amiodarone (0.01, 0.1, 1, 10, 100 micromol/L) for 3 hours or amiodaron (10 micromol/L) for 0, 0.5, 1, 3, 6 hours. The I(f) and HCN 1 - 4 gene expressions were measured through the whole-cell configuration of the patch-clamp technique and real-time quantitative polymerase chain reaction (real-time PCR) using SYBR Green PCR kit., Results: (1) HCN1, HCN2, HCN3 and HCN4 represented (0.23 +/- 0.01)%, (83.58 +/- 0.04)%, (0.79 +/- 0.01)% and (15.44 +/- 0.01)% of total HCN mRNA, respectively. (2) Amiodaron resulted in a dose-dependent I(f) [(3.1 +/- 0.9)%, (9.7 +/- 2.4)%, (36.7 +/- 5.8)%, (80.3 +/- 1.8)% and (85.9 +/- 3.1)%, respectively at -145 mV, IC(50) (1.32 +/- 0.28) micromol/L], HCN2 [(2.1 +/- 0.8)%, (8.9 +/- 3.6)%, (30.1 +/- 4.2)%, (78.3 +/- 3.6)% and (81.1 +/- 1.9)%, respectively] and HCN4 decrease [(0.5 +/- 0.2)%, (2.1 +/- 2.6)%, (8.8 +/- 3.2)%, (60.1 +/- 4.6)% and (59.6 +/- 6.5)%, respectively]. (3) Amiodaron (10 micromol/L) also induced a time-dependent I(f) [(1.1 +/- 0.1)%, (12.6 +/- 2.3)%, (80.6 +/- 2.2)% and (80.1 +/- 2.1)%, respectively], HCN2 [(1.0 +/- 0.1)%, (9.8 +/- 3.9)%, (82.9 +/- 4.6)% and (83.9 +/- 1.7)%, respectively] and HCN4 decrease [(0.1 +/- 0.1)%, (1.9 +/- 1.1)%, (59.4 +/- 7.8)% and (60.9 +/- 3.1)%, respectively]. However, HCN1 and HCN3 expressions were not affected by amiodaron treatment., Conclusion: Current density of I(f) and the expression of HCN2 and HCN4 were decreased by amiodaron which might be the possible antiarrhythmic working mechanisms of amiodaron.

Aim: To construct and screen the specific Fab phage antibody library against human Raji cell strain in B-lymphoma., Methods: BALB/c mice were immunized with Raji cells, and the antibody light chain kappa genes and heavy chain genes Fd from the spleen cells were amplified by RT-PCR. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the light chain kappa genes and heavy chain genes Fd were inserted into the phagemid vector pComb3H-SS successively and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Raji cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. The specific antibodies against Raji cells were obtained after selected with Raji cells. The binding activity with antigens was identified by ELISA and the positive clones were sequenced., Results: The Fab phage antibody library with 2.18 x 10(7) volume was constructed and eight positive clones which specifically recognized Raji cell strain were isolated. Sequence analysis of the two positive clones showed that the variable heavy domains (VH) and variable light domains (VL) were highly homologous with the registered murine Ig heavy chain V region sequences and kappa light chain sequences, respectively., Conclusion: Fab phage antibody library was successfully constructed and specific antibodies against membrane antigens in Raji cells were obtained, which will provide an experimental foundation for the further investigation of B-lymphoma immunotherapy.

Background: Hypertrophic cardiomyopathy (HCM) is a form of cardiomyopathy with an autosomal dominant inherited disease, which is caused by mutations in at least one of the sarcomeric protein genes. Mutations in the beta-myosin heavy chain (beta-MHC) are the most common cause of HCM. This study was to reveal the disease-causing gene mutations in Chinese population with HCM, and to analyze the correlation between the genotype and phenotype., Methods: The exons 3 to 26 of MYH7 were amplified by PCR, and the PCR products were sequenced in five non-kin HCM patients. A 17-year-old patient was detected to be an Arg723Gly mutation carrier. Then his family was gene-screened, and the correlation between genotype and phenotype was analyzed., Results: The mutation of Arg723Gly in a Chinese family with HCM was detected for the first time. With a C-G transversion in nucleotide 13,619 of the MYH7 gene, located at the essential light chain interacting region in S1, the replacement of arginine by glycine took place at amino acid residue 723. A two-dimensional echocardiogram showed moderate asymmetrical septal hypertrophy with left atria enlargement. There was no obstruction in the left ventricular outflow tract. In his family, a total of 13 individuals were diagnosed HCM and 5 of them were dead of congestive heart failure at a mean age of 66-year-old. Eight living members were all detected to carry the mutation, in which 3 developed progressive heart failure. Moreover, the heart function of the people evidently deteriorates when their age are older than 50. The mutation and the disease show co-separated., Conclusion: The Arg723Gly mutation is a malignant type. In Chinese the mutation has the similar characters to the former report but has low degree malignant.

Objective: To study on the first Chinese congenital thrombotic thrombocytopenic purpura (TTP) patient with respect to peripheral blood platelet count and ADAMTS13 activity, ADAMTS13 antigen, anti-endothelial cell antibody (ACEA), and thrombospondin1 (TSP1) etc. to compared it with idiopathic TTP and to explore something special and meaningful in the pathogenesis of congenital TTP., Methods: A total of 30 volunteers served as controls. The congenital TTP patient and 10 carriers in her pedigree as well as 7 idiopathic TTP patients were included in this study. Residue collagen binding assay and a newly developed sandwich ELISA were used for determination of ADAMTS13 activity and antigen respectively. An ELISA for ACEA and a commercial kit for TSP1 were use in the study., Results: After a "3-4 weeks regular relapse" of the congenital TTP, which is similar to that in other reports, the Chinese patient moved into a new relapse cycle. The average of the antigen levels of this patient before plasma exchange (PE) and at the interval between relapses was (22.79 +/- 14.61) U/L (P < 0.01), while that of Chinese normal control (NC) turned out to be (600.93 +/- 145.36) U/L. TSP1 detected at the same time (4.67 +/- 1.62) mg/L in the congenital TTP patient was much lower than that in NC (18.34 +/- 7.24) mg/L (P < 0.01). A value in ACEA assay was 0.58 +/- 0.06 (P < 0.01) for congenital TTP, which was a little higher than that in NC (0.40 +/- 0.13)., Conclusions: Congenital TTP may have changeable relapse cycles during episodes. ADAMTS13 antigen and TSP1 and ACEA in congenital TTP showed significant difference as compared with those in NC. ADAMTS13 and ACEA in congenital TTP were also markedly lower than those in idiopathic TTP.

Objective: To explore the biological effect on NB4 cells proliferation, all-trans retinoic acid (ATRA) inducing differentiation and resistance to apoptosis by vascular endothelial growth factor (VEGF)-C cDNA transfection., Methods: The recombinant eukaryotic expression plasmid pcDNA3.1-VEGF-C and the vacant pcDNA3.1 vector were introduced separately into NB4 cells by lipofectamine mediation. The positive clones were screened by G418 and identified by reverse transcriptase-PCR (RT-PCR) and Western blotting. The proliferation capacity of NB4/VEGF-C cells was analysed by MTT assay and colony forming assay in vitro. After NB4/VEGF-C cells were induced by ATRA, the expression level of C/EBPalpha gene, CD11b on cells surface and morphological alteration were analysed by real-time quantitative PCR (RQ-PCR), flow cytometry (FCM), and Wright-Giemsa staining, respectively. FCM Annexin V-FITC/PI dual labeling technique was performed to investigate the etoposide (Vp16) induced NB4/VEGF-C cells apoptosis and bcl-2 gene expression level in these cells was analysed by RQ-PCR. The NB4/pcDNA3.1 cells was used as control in the above experiments., Results: A stable NB4 cell line that secrets VEGF-C and its control lines were established. The proliferation capacity of the former was stronger than that of the latter. The expression level of C/EBPalpha gene of NB4/VEGF-C cells on ATRA treatment was only 1/32 that of NB4/pcDNA3.1 cells. The CD11b level and the degree of differentiation of NB4/VEGF-C were weaker than that of NB4/pcDNA3.1 cells. The percentage of apoptotic NB4/VEGF-C cells induced by Vp16 [(7.20 +/- 2.52)%] was significantly lower than that of NB4/pcDNA3.1 cells [(16.07 +/- 3.58)%] (P = 0.005), but the bcl-2 gene expression level of NB4/VEGF-C cells is 2.28-fold that of NB4/pcDNA3.1 cells., Conclusion: The VEGF-C via VEGFR-3 signaling pathway could promote the proliferation of leukemic cells by autocrine pathway and inhibit the cell differentiation mediated by ATRA and chemotherapy-induced apoptosis. VEGF-C/VEGFR-3 signaling loops might play an important role in disease progression and be potential therapeutic target for the treatment of leukemias.

Objective: Hypertrophic cardiomyopathy (HCM) is a genetically and phenotypically heterogeneous disease and an Arg723Gly mutation in beta-myosin heavy chain (beta-MHC) gene was found in 3 Spanish families with malignant HCM. We detected this gene mutation in 5 Chinese pedigrees with hypertensive cardiomyopathy., Methods: Five Chinese pedigrees with HCM and 80 age-matched normal control subjects were chosen for the study. The exons in the functional regions of the beta-MHC gene were amplified with PCR and the products were sequenced, genotype and phenotype analyzed., Results: Arg723Gly mutation was identified in exon 20 in one pedigree. In this pedigree, 13 out of 25 family members were diagnosed as HCM, 5 died of heart failure, all HCM patients in this pedigree had Arg723Gly mutation and 3 of them had NYHA III and 2 of them were diagnosed as HCM before the age of 20., Conclusions: Arg723Gly mutation was also one of the main disease-causing genes in Chinese familial HCM. The mutation of Arg723Gly is a malignant phenotype as shown by early progressive heart failure development and poor prognosis in this pedigree with Arg723Gly mutation.

Objective: To investigate the antigen levels and activity of von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura (TTP) patients and carriers., Methods: 28 samples from 13 TTP patients and 10 samples from the carriers were examined. The activity of ADAMTS13 was measured by residue collagen binding assay, and antigen by a newly developed sandwich ELISA., Results: The mean ADAMTS13 level in Chinese normal controls (CN) was (600.93 +/- 145.36) mU/ml (n = 26) comparable to the level (1000 mU/ml) in pooled normal Caucasian plasma, and the activity was (74.79 +/- 11.81)%. Both the antigen level and activity of ADAMTS13 in congenital TTP patients either before plasma exchange (pre-PE) or interval relapse were quite lower than those in normal control, but were increased after PE (post-PE). The antigen was (331.40 +/- 109.85) mU/ml (P < 0.01, n = 10), and activity was (66.79 +/- 12.82)% (P > 0.05). The ADAMTS13 levels pre-PE in idiopathic TTP was (98.7 +/- 82.08) mU/ml (n = 11, P < 0.01), and that post-PE was up to (449.4 +/- 232.33) mU/ml (P < 0.01, n = 10). The activity of ADAMTS13 in patients pre-PE and post-PE were (22.23 +/- 19.07)% (P < 0.01) and (60.92 +/- 22.33)% (P > 0.05) respectively. In a secondary TTP patient the ADAMTS13 antigen was much higher than that in CN, and the activity was 6.00%., Conclusion: The antigen and activity of ADAMTS13 in most TTP patients pre-PE are deficient, and these two indices in most TTP patients are paralleled. The reason for ADAMTS13 deficiency is congenital shortage or clearance by immune system, but it is unknown that why in some patients the ADAMTS13 antigen is extremely high but its activity is quite low.

Objective: To identify the genetic defect of inherited coagulation factor (F) deficiency in a Chinese family and to explore its molecular mechanism., Methods: The activity and antigen of plasma F were measured by photometric test and enzyme-linked immunosorbent assay, and rocket-electrophoresis, respectively. All the exons and exon-intron boundaries of the FA subunit gene were amplified by PCR and then DNA sequencing was performed. Restriction endonuclease analysis was used for the PCR products of the family members and 80 healthy donors to exclude gene polymorphism., Results: Rapid dissolution of the proband's fibrin clot occurred within 30 minutes, and antigen of his plasma F was significantly decreased, two compound heterozygous missense mutations (a C to T transition at nucleotide 177,246 which caused Arg703Trp, and a A to G transition at nucleotide 177,286 which caused His716Arg) in exon 15 of FA subunit gene were found. The possibility of gene polymorphism was excluded by restriction endonuclease analysing. Each of these two missense mutations was respectively found in his mother and father. Molecular modeling based on 3D crystallographic data predicted that the mutant protein decreased stability and was likely to be rapidly degraded., Conclusions: The inherited F deficiency in the Chinese family is caused by two compound heterozygous missense mutations-Arg703Trp and His716Arg in the FA subunit, which to our knowledge, are reported for the first time.

Objective: To measure plasma thrombospondin1 (TSP1) in thrombotic thrombocytopenic purpura (TTP) and other diseases such as idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), myocardial infarction, brain infarction, and malignant tumor et al. and 8 patients after bone marrow transplantation (BMT) were also investigated. Then to study on the relationship between TSP1 and von Willebrand factor cleaving protease (ADAMTS13); and to identify the significance of plasma TSP1 in TTP., Methods: TSP1 was measured by a commercial kit and the activity of ADAMTS13 was evaluated by residue collagen binding assay., Results: TSP1 in TTP plasma before plasma exchange or plasma infusion was 6.49 mg/L, and stepping up to 13.02 mg/L after therapy, but still significantly lower than 18.34 mg/L in normal control. While the decrease in different degree of ADAMTS13 activity was observed from 0%-52%, and there were 8 samples whose activity of ADAMTS13 were no more than 10%; the different extent of increase in those patients after therapy was demonstrated to be 2.9%-93.4%, only one patient's ADAMTS13 activity was below 10%. The activity of ADAMTS13 in some ITP and SLE patients were mildly decreased (63% +/- 16% and 70% +/- 14% respectively), TSP1 were also decreased (16 mg/L +/- 8 mg/L). TSP1 in patients of myocardial infarction and brain infarction were increased (24.0 mg/L +/- 2.9 mg/L), while ADAMTS13 activity had no significant change (72% +/- 16%). Same things happened in BMT patients., Conclusion: There was some concordance between the decrease of ADAMTS13 activity and TSP1 in plasma of TTP patients. And the change of TSP1 was restricted to TTP. TSP1 may contribute to the episode of TTP in a still unclear fashions.

Background & Objective: Vascular endothelial growth factor-C/vascular endothelial growth factor receptor-2, -3 (VEGF-C/VEGFR-2,3) signaling pathway can induce angiogenesis/lymphangiogenesis, and promote invasion and metastasis of solid tumors. Recent studies showed that VEGF-C and its receptors also expressed on hematological tumor cells, but their effects on leukemic cells are unclear. This study was to explore the effects of recombinant VEGF-C secreted from VEGF-C cDNA-transfected CHO (CHO/VEGF-C) cells on proliferation and chemotherapy-induced apoptosis of leukemic cells., Methods: The effect of recombinant VEGF-C (in the supernatants of CHO/VEGF-C cells) on angiogenesis was observed using the chicken chorioallantoic membrane model. The expression of VEGFR-2 and VEGFR-3 on leukemic cell lines TF-1, K562, HEL, NB4, HL-60, MR2, U937,SHI-1, and RPMI8226 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). The effect of VEGF-C on the proliferation of NB4 and K562 cells was assessed by MTT assay; its effect on the apoptosis of NB4 cells, which were treated with chemotherapeutic agents [etoposide (VP-16), daunorubicin (DNR), or arsenic trioxide (As2O3)], was investigated by FCM with Annexin V/PI double staining. The supernatant of CHO/pcDNA3.1 cells was used as control., Results: The recombinant VEGF-C induced angiogenesis in the chicken chorioallantoic membrane model. VEGFR-3 was expressed in NB4, HEL, and RPMI8226 cells; VEGFR-2 was expressed in HEL and TF-1 cells. Compared with the controls, the recombinant VEGF-C promoted the proliferation of NB4 cells (VEGFR-3(+)) (P(24h)=0.006, P(48h)=0.018), but had no effect on K562 cells (VEGFR-3(-)); it inhibited the chemotherapy-induced apoptosis of NB4 cells (P(VP-16)=0.019, P(DNR)=0.013, P(As2O3)=0.042)., Conclusion: The recombinant VEGF-C could induce angiogenesis, and may promote the proliferation and inhibit the chemotherapy-induced apoptosis of leukemic cells via VEGFR-3 signaling pathway, which might be potential target therapy for leukemia.

Objective: To investigate the gene mutations of ADAMTS13 in a highly suspected hereditary thrombocytopenic purpura (TTP) patient, and then make a progressive diagnosis and adjust the plan of therapy., Methods: ADAMTS13 activity and inhibitor were determined by residual-collagen binding assay during several episodes. Genomic DNA extracted from the proband's peripheral blood was used for amplification of 29 exons and exon/intron boundaries of ADAMTS13 by PCR. The PCR products were screened by direct sequencing and the gene alterations were further confirmed by direct sequencing in her family members., Result: The activity of the proband's ADAMTS13 was significantly reduced while no inhibitor was found. Two novel missense mutations were found in the TSPI repeated motif domain of ADAMTS13. In both mutations, thymine substituted for cytidine, resulting in the substitution of leucine for serine in nt 2708, exon 21 (codon S903L), and tryptophan for arginine in nt 3283, exon 25(codon R1095W). These two mutations were revealed as each heterozygote in the proband's parents., Conclusion: The deficiency of ADAMTS13 caused by two homozygote missense mutations might be responsible for episode of this TTP patient.

This study was aimed to further investigate the function of platelet collagen receptor-glycoprotein VI and to screen its specific inhibitor. The extracellular domain of platelet glycoprotein VI (GPVI) in E. coli was expressed by recombinant technology, the extracellular domain cDNA of GPVI was amplified from pBluescript KS(-)-GPVI plasmid by PCR. Proved by sequencing, the expression vector pET-20b(+)-GPVI was constructed, which was then transformed into E. coli (BL21(DE3)pLysS) and induced by IPTG. The recombinant GPVI was purified on Ni-NTA resin column and renatured in PBS containing GSH and GSSG. The anti-penta His McAb and anti-GPVI polyclonal antibody were used to identify the recombinant GPVI in Western blotting. Collagen binding test was conducted to investigate the biological activity of recombinant GPVI. The results showed that the recombinant GPVI was expressed in E. coli and successfully purified, which was confirmed to be similar to the native GPVI in Western blotting. The recombinant GPVI can bind the type I collagen in dose-dependent manner. In conclusion, the recombinant GPVI can be achieved in E. coli and restore its native characteristics after renaturation.

Objective: To identify the genetic defect underlying congenital afibrinogenemia in a Chinese family., Methods: Plasma fibrinogen (Fg) was assessed by both Clauss method and immunonephelometry. Genomic DNA was isolated from peripheral blood of the proband and 13 members of her family. All the exons and exon-intron boundaries of the three fibrinogen genes (FGA, FGB, FGG) were amplified by PCR followed by direct sequencing. Restriction endonuclease analysis was performed for the PCR products of the family members and 50 healthy donors to exclude gene polymorphism., Results: No Fg was detected in the plasma of the proband and her father by Clauss method, while low levels (< 0.02 g/L) were detected by immunonephelometry. A homozygous C to T mutation was found in the two cases at nucleotide 3108 in exon 4 of FGA gene, resulting in a null mutation which encoded severely truncated alpha-chains owing to its premature termination at the Gln 150 codon. The C-->T mutation eliminated a unique recognition site for restriction enzyme RsaI. The PCR amplified fragments of the proband and her father could not be digested by RsaI, showing that they are homozygous. Her mother and some family members are heterozygous at this site since the fragment could partly be digested, while the same fragment of controls could be completely digested as expected., Conclusion: The Gln (CAG)-->150stop (TAG) nonsense mutation in FGA gene is a novel genetic defect of congenital afibrinogenemia which, to our knowledge, has not been described before.

Objective: To investigate the T13254C polymorphism frequency in GPVI gene among Chinese Han population and its relevance to the arterial thrombotic diseases., Methods: The enrolled population in this study consisted of 314 healthy subjects and 274 patients with myocardial or cerebral infarctions. GPVI T13254C genotypes were determined by PCR amplification of a 355 bp fragment encompassing exon 5 of GPVI gene, followed by Msp I digestion of the product. The digested products were analyzed in 15% polyacrylamide gel electrophoresis (PAGE)., Results: The frequencies of the T allele and C allele in the T13254C polymorphism were 0.9809 and 0.0191, respectively, with a frequency of heterozygous of 0.0319, which were significantly different from those reported in western population (P < 0.01). As compared with controls, no significant difference in T13254C genotype distribution was found in the arterial thrombotic diseases group., Conclusion: The GPVI T13254C polymorphism appears in a low frequency in Chinese Han population. No relationship is found between T13254C polymorphism and the risk for thrombotic diseases.

The patient was a 34-year-old man with life-long bleeding episodes, whose hemorrhage problem was characterized predominantly by prolonged bleeding at surgical or traumatic sites. All routine coagulation parameters were within normal ranges. The patient's bleeding tendency was not caused by factor XIII deficiency, alpha2-antiplasmin deficiency, or tissue type-plasminogen activator increase. His characteristic abnormalities of fibrinolysis included shortened euglobulin clot lysis time, low plasminogen activator inhibitor-1 (PAI-1) activity and antigen in plasma, which were remarkably reduced to only about 10% of control. An operation was performed in order to clear two hematomas in the patient's left leg and hip, and subsequent bleeding episodes were well controlled with adjuvant administration of intravenous aminomethylbenoic acid after surgery. PAI-1 gene analysis by polymerase chain reaction product sequencing revealed that the patient had a heterozygous missense mutation G to A transition at nucleotide position 4497 in exon 2, causing replacement of alanine 15 (GCC) to threonine (ACC) at signal peptide. The restriction endonuclease analysis showed that this gene mutation also existed in the patient's father, but not in his mother and 60 normal subjects. The wild-type and mutant plasmids were constructed and transiently transfected into Chinese hamster ovary cell lines; the levels of PAI-1 activity and antigen in the media of the mutant were approximately 70% of the wild type, and the levels of PAI-1 protein in cell lysates were almost equal in wild-type and mutant plasmids. These results indicate that the mutation in signal peptide may partly impair the secretion of PAI-1.

Objective: To report a patient with congenital plasminogen activator inhibitor-1 (PAI-1) deficiency and explore its molecular mechanism., Methods: The activities of tissue plasminogen activator (tPA), alpha(2) antiplasmin (alpha(2)AP) and PAI-1 were measured by the methods of chromogenic substrate, the antigens of tPA and PAI-1 were measured by ELISA. PAI-1 gene was studied by PCR product sequencing and restriction endonuclease ana-lysing., Results: In the present patient, the euglobulin clot lysis time was 70 minutes and was corrected to normal range after added 50 ng/ml PAI-1 to his plasma. The activities of t-PA, alpha(2)AP, and factor were normal; the activity and antigen of PAI-1 in plasma were both significantly decreased. Nucleotide sequence analysis revealed that the patient had a heterozygous missense mutation in exon 2, a G to A transition at nucleotide 43. The possibility of gene polymorphism was excluded by restriction endonuclease analysing., Conclusions: It is the first patient with congenital PAI-1 deficiency reported in China. The PAI-1 deficiency in the patient may be caused by compound heterozygosity, one of which is the G to A transition at nt43, a new mutation in congenital PAI-1 deficiency.

Aim: To study whether securinine might induce apoptosis in human leukemia HL-60 cells., Methods: Inhibition of proliferation was measured using MTT assay. The amount of apoptotic cells was measured by flow cytometry. DNA fragmentation was visualized by DNA agarose gel electrophoresis and the cellular changes were observed by electron microscope., Results: Securinine 5-80 mg.L-1 elicited typical apoptosis morphological changes and DNA fragmentation in a concentration-dependent manner in HL-60 cells. Securinine inhibited HL-60 cell proliferation in a time- and concentration-dependent manner. The IC50 and 95% confidence limits were 27 (15-47) mg.L-1 after 12-h treatment with securinine., Conclusion: Securinine induced apoptosis in HL-60 cells.