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To investigate the clinical characteristics and prognosis of patients with medium and high risk myelodysplastic syndrome (MDS).97 MDS patients above the age of 60 treated in Nanfang Hospital, Southern Medical University from February 2011 to August 2020 were enrolled. The clinical characteristics and prognosis of the MDS patients with medium risk, high risk or very high risk based on IPSS-R category were retrospectively analyzed. According to the difference of treatment regimes, the patients were divided into the transplantation group, chemotherapy group and other treatment group, and the efficacy among the patients in the 3 groups were analyzed.MDS with excess blast (MDS-EB) in the elderly patients with medium and high risk MDS were the most common, 47.4% of the patients with abnormal chromosome karyotypes, and 23.7% with complex karyotypes (≥3). 97.3% of the patients showed at least one gene mutation, and TP53 mutations were detected in nearly 20% of the patients with medium and high risk. Multivariate analysis showed that IPSS-R category and treatment regimes were the factors affecting the prognosis of elderly patients with medium and high risk MDS. The median overall survival (OS) time of the patients in the 3 groups showed significant difference (P=0.012), and the median OS of the patients in the transplantation group was significantly longer than that in the chemotherapy group and other group (P=0.003,P=0.014,respectively), while there was no significant difference in median OS between chemotherapy group and other treatment group (P=0.685).Elderly MDS patients with medium and high risk can benefit from allogeneic hematopoietic stem cell transplantation, which will prolong their OS.老年中高危骨髓增生异常综合征患者的临床特点及预后研究.探讨老年中高危骨髓增生异常综合征（MDS）患者的临床特点及预后.回顾性分析2011年2月至2020年8月在南方医科大学南方医院就诊的97例年龄≥60岁、IPSS-R危险分层为中危/高危/极高危MDS患者的临床特点及预后情况。根据采取治疗方案的不同，将患者分为移植、化疗和其他治疗3组，分析评估不同治疗组患者之间疗效的差别.老年中高危MDS患者以MDS伴原始细胞增多型（MDS-EB）为主，47.4%的患者合并染色体核型异常，其中复杂核型（≥3个）占23.7%。97.3%的患者至少存在一种基因突变，TP53突变在老年中高危MDS患者中检出率近20%。多因素分析显示，IPSS-R危险分层、治疗方案影响老年中高危MDS患者的预后。3组患者的中位生存（OS）时间统计学差异（P=0.012），移植组中位OS长于化疗组及其他治疗组（P=0.003、P=0.014），化疗组和其他治疗组的中位OS无统计学差异（P=0.685）.老年中高危MDS患者可从异基因造血干细胞移植中获益，OS延长.

To investigate the correlation between U2AF1 gene mutation and clinical manifestations and prognosis in patients with myelodysplastic syndromes (MDS).The clinical data of 203 MDS patients who accepted Next Generation Sequencing (NGS) was retrospectively analyzed in Nanfang Hospital, Southern Medical University from December 2012 to October 2019. According to whether the patients had U2AF1 gene mutation, the patients were divided into U2AF1 mutated group and non-mutated group, and the relationship between gene mutation characteristics and clinical manifestations and prognosis was analyzed. Then according to the difference of the mutation site of U2AF1, the patients in U2AF1 mutated group were divided into U2AF1The incidence of U2AF1 mutation in MDS patients was approximately 11.3% (23/203), and the mutation frequency of U2AF1 allele was 32.5%. The male ratio in U2AF1 mutated group was significantly higher than that in U2AF1 non-mutated group (P=0.001). There was no patient who had complex karyotypes or TP53 gene mutation in U2AF1 mutated group. There were no significant differences in ages, blood parameters, bone marrow blasts, WHO 2016 classification, IPSS-R category, chromosomal abnormalities like del(5q), -7/del(7q), del(20q), +8, and gene mutation like ASXL1, DNMT3A, RUNX1, SF3B1, and SRSF2 mutation between U2AF1 mutated group and the non-mutated group. Compared with the non-mutated group, there was no significant difference in the overall survival time (P=0.377), the time of acute myeloid leukemia (AML) transformation (P=0.681), and the response rate to hypome- thylating agents in U2AF1 mutated group (P=0.556). Besides, no differences were observed in sex, diagnosis age, WHO 2016 classification, IPSS-R category, blood parameters, overall survival time, and AML transformation time between U2AF1The U2AF1 gene mutation dose not affect the survival time, AML transformation time, and response rate to hypomethylating agents in MDS patients. Besides, there are no statistical differences in the clinical characteristics and prognosis of MDS patients between U2AF1骨髓增生异常综合征患者U2AF1基因突变特征与临床表现及预后的相关性.探究骨髓增生异常综合征（MDS）患者U2AF1基因突变特征与临床表现及预后的相关性.回顾性分析2012年12月至2019年10月在南方医科大学南方医院进行二代测序（NGS）检测的203例MDS患者资料。根据患者有无U2AF1基因突变，将患者分为U2AF1突变组和U2AF1非突变组，并对2组患者的基因突变特征与临床表现及预后关系进行了分析；根据U2AF1突变位点，分为U2AF111.3%（23/203）的MDS患者存在U2AF1突变，U2AF1等位基因突变频率为32.5%。U2AF1突变组男性比例明显高于U2AF1非突变组（P=0.001）。U2AF1突变组无1例患者为染色体复杂核型或TP53突变。2组患者年龄、血液学参数、骨髓原始细胞比例、WHO 2016分型、IPSS-R危险分层、5q-、 -7/7q-、20q-、+8等染色体异常及ASXL1、DNMT3A、RUNX1、SF3B1、SRSF2等基因突变差异均无统计学意义。与U2AF1非突变组相比，U2AF1突变组患者的生存时间（P=0.377）、转化为白血病时间（P=0.681）及对去甲基化药物的总反应率（P=0.556）差异均无统计学意义。此外，U2AF1本研究结果提示，U2AF1基因突变不影响MDS患者总生存时间、转化为白血病率以及对去甲基化药物的反应率。U2AF1

To investigate whether cell preservation solution can prolong the survival time of leukemia cells and increase the survival rate, so as to improve the detection rate of central nervous system leukemia.Kasumi cells were added into cerebrospinal fluid (CSF) supernatant with or without cell preservation solution to compare cell viability and biological characteristics at different time point. Wright Giemsa staining was used to compare cell morphology; cell counting, CCK-8 method, and trypan blue staining were used to compare the cell number, and flow cytometry was used to compare the cell viability. The expression of AML-ETO tumor fusion gene was detected by fluorescence quantitative RT-PCR.At different time points (8 h and 24 h), the survival, molecular biological characteristics and RT-PCR result of the cells in CSF with cell preservation solution were significantly better than those in normal cerebrospinal fluid.Cell preservation solution can effectively improve the survival time and survival rate of leukemic cells, thereby increase the detection rate of CNS leukemia.

A comparison experiment has been taken in the Kiaochow Bay between a newly designed mooring turbulence observation instrument (MTOI) and microstructure profiler MSS60 made by Sea & Sun. The whole observing system is based on a submerged buoy, in which the turbulence observation instrument is embedded, with a streamline-shape floating body, which is made of buoyancy material of glass microsphere. For the movement of seawater and the cable shaking strongly anytime influence the behaviors of the floating body, the accelerate sensors are used for the vibration measurement in the instrument together with the shear probe sensor. Both the vibration data and the shear data are acquired by the instrument at the same time. During data processing, the vibration signals can be removed and leave the shear data which we really need. In order to prove the reliability of the new turbulence instrument MTOI, a comparison experiment was designed. The measuring conditions are the same both in time and space. By this way, the two groups of data are comparable. In this paper, the conclusion gives a good similarity of 0.93 for the two groups of shear data in dissipation rate. The processing of the data acquired by MTOI is based on the cross-spectrum analysis, and the dissipation rate of it matches the Nasmyth spectrum well.

To investigate the incidence and risk factors of sclerodermatous chronic graft-versus-host disease (ScGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).The clinical data of 259 patients undergoing allo-HSCT in Nanfang Hospital between January, 2012 and December, 2014 were analyzed.Chronic GVHD following allo-HSCT occurred in 134 (51.7%) cases, among whom 22 patients showed sclerodermatous features at a median of 12.5 months (range 4-28 months) after the transplantation. The overall incidence of ScGVHD was 8.49% (22/259) in the recipients and 16.4% (22/134) in those with cGVHD. Univariate analysis showed that the conditioning regimen with total body irradiation (P=0.031), GVHD prophylaxis with MMF (P=0.046), presence of chronic GVHD (P=0.008), and donor lymphocyte infusion (P=0.001) were all closely associated with the occurrence of ScGVHD. Multivariate analysis identified chronic GVHD (RR=3.512, 95%CI: 1.235-9.987, P=0.018) and donor lymphocyte infusion (RR=5.217, 95%CI: 1.698-16.029, P=0.004) as the independent risk factors of ScGVHD.ScGVHD following allo-HSCT is not a common complication, and cGVHD and donor lymphocyte infusion are the independent risk factors for ScGVHD.

To establish allo-transplantation model by using mRFP⁺ to eGFP⁺ transgenic mice and to observe the distribution of donor cells and donor-recipient cellular interaction in the bone marrow after semi-solid decalcification (SSD).After myeloablative irradiation, C57BL/6 female eGFP⁺ transgenic mice were infused with (5 × 10⁶) bone marrow cells from FVB male donor mice through tail vein. The control group was infused with PBS. Then the general conditions, engraftment level, hematopoietic recovery, incidence of GVHD and survival of recipients were evaluated after transplantation. In the recovery process, SSD was used to treat the femora before observing the cells distribution, morphology and interaction by confocal microscopy directly or after making frozen section.WBC of recipient eGFP⁺ mice was recovered on (20 ± 3.07) d, (93.94 ± 1.59)% in peripheral cells were RFP⁺ cells (n = 10), GVHD happened in 4 of 10 mice within 1 month. During SSD, the hard components were replaced gradually and RFP⁺ cells could be seen mainly in the bone trabecula and surrounded by eGFP⁺ cells under confocal microscope, their interactions could be further observed clearly in bone marrow microenvironment in three-dimensional reconstruction.The double fluorescent allo-transplantation mouse model successfully established, by means of our novel protocol named SSD, the donor and recipient cell location and their interaction can be visually observed, which provides the basis for clinical studies on the distribution and homing of donor cells, and some related explorations after transplantation.

The study was to analyze the acute heart failure's risk factors and clinical characteristics for the patient with chronic myelogenous leukemia (CML) during the early stage (within 100 d) of allogeneic hematopoietic stem cell transplantation (allo-HSCT).A total of 106 cases of CML received allo-HSCT were retrospectively studied in Nanfang Hospital from May 2003 to May 2013. On the basis of existence or absence of acute heart failure during early stage of allo-HSCT (100 d), the patients were divided into heart failure (15 cases) and control group (91 cases). Using Logistic univariate analysis, Fisher' exact test and Pearson X(2) test, the acute heart failure's risk factors and clinical characteristics of both groups were analyzed.The median occurrence time of acute heart failure was 3 d (1 d before transplantation to 84 d after transplantation). Logistic univariate analysis indicated that the imatinib treatment history and time, and the prophylaxis regimens for GVHD with anti-thymocyte globulin (ATG) were all the poor prognostic factors for acute heart failure. Incidence of hepatic veno-occlusive disease (HVOD), bacterial infection and adverse prognostic events including death in the heart failure group patients were statistically higher than that in control group (P0.05).Acute heart failure mostly happened in the early stage after allo-HSCT, imatinib treatment and GVHD prophylaxis regimens with ATG are the poor prognostic factors for acute heart failure. The patients of heart failure group seem to have higher incidence of hepatic veno-occlusive disease (HVOD), bacterial infection and deaths.

PCR is an enabling and preferred technology for the detection of DNA or mRNA in genomic age, and has been widely used in molecular medicine, biotechnology, microbiology and diagnostics. PCR has many excellent traits, such as high specificity and sensitivity, short handle time, reliable and effective results, low demand for sample. In recent years, real-time reverse transcription PCR (qRT-PCR) has acquired an impressive improvement in the detection of mRNA, it has a perfect sensitivity and can quantify some rare mRNA copies as low as less ten. The progress in PCR results in a more strict demand for sample, so it is necessary to take the potential PCR-inhibitory components in peripheral blood and bone marrow samples into account again. This review explores the potential PCR-inhibitory components in peripheral blood and bone marrow samples, and discusses the possible mechanism and solutions for the inhibition effect, also emphasizes the potent effect of the frequently-used anticoagulant heparin sodium in PCR. This article will be helpful to assess the PCR detection results (especially qRT-PCR ) in some degree, for example, can use it to decide whether the PCR detection results are reasonable for some low-expression genes, or use it to eliminate the false negative results generated by PCR-inhibitory components in sample.

The accuracy and robustness of x-corners detection are critical in camera calibration. In order to achieve high accuracy and robust performance in blurred and heavily distorted images automatically, the Martin Rufli’s algorithm is improved. The Martin Rufli’s algorithm is robust to detect x-corners in blurred and heavily distorted images; however, the x-corners detected are not accurate enough, and so we take the center of gravity, which is weighted by the squared gradients in order to refine x-corners. As gradient represents grayscale change between a pixel and its adjacent pixels, more accurate x-corners can be detected. Compared with the method proposed by Martin Rufli, experiment results indicate that the improved algorithm can improve accuracy capacity.

This study was aimed to assess the effect of Astragalus Polysaccharide (ASPS) on in-vitro hematopoiesis. CFU-GM assays were used to determine the effect of ASPS and thrombopoietin (TPO) on granulocytic-monocyte progenitor cells. The CFU assays were also used to investigate the effect of ASPS on the proliferation of HL-60 cells.HL-60 cells were cultured with serum-free RPMI 1640 medium and treated with or without of different concentrations of ASPS. After 72 h incubation, the number of cells were counted.In addition, the caspase-3 and JC-1 expression was determined by flow cytometry with Annexin V/PI double staining. The results showed that ASPS (100, 200 µg/ml) and TPO (100 ng/ml) significantly promoted CFU-GM formation in vitro. Various concentrations of ASPS and TPO also promoted the colony formation of HL-60 cells, the largest effect of ASPS was observed at a concentration of 100 µg/ml. There were no synergistic effects between TPO and ASPS on cellular proliferation. The results also showed that ASPS significantly protected HL-60 cells from apoptosis in condition of serum-free medium culture, suppressed caspase 3 activation, and reduced the cell apoptosis. It is concluded that ASPS can significantly promote the formation of bone marrow CFU-GM and the proliferation of HL-60 cells, the optimal concentration of ASPS is at 100 µg/ml. In the absence of serum inducing apoptosis, ASPS also significantly reduced the apoptosis of HL-60 cells via suppressing the activation of caspase-3.

To compare the transplant-related toxicity and the efficacy of busulfan/fludarabine (Bu/Flu) and busulfan/cyclophosphamide (Bu/Cy) as conditioning regimen in allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute myeloid leukemia(AML) in the first complete remission (CR1).Totally 32 AML-CR1 patients underwent allo-HSCT were divided into Bu/Cy (Bu 3.2 mg×kg(-1)×d(-1), 7 - 4 days before transplantation; Cy 60 mg×kg(-1)×d(-1), 3 - 2 days before transplantation) and Bu/Flu (Bu 3.2 mg×kg(-1)×d(-1), 5 - 2 days before transplantation; Flu 30 mg×m(-2)×d(-1), 6 - 2 days before transplantation) groups. The regimen-related toxicity (RRT), incidence and severity of graft-versus-host disease (GVHD), 3-year cumulative relapse rate, non-relapse mortality (NRM), 3-year event-free survival (EFS) rate and overall survival (OS) rate were compared between the two groups.The median follow-up duration was 617.5 (6 - 1261) days. All patients achieved successful engraftment on 30 day after transplantation. There were no significant differences in the median time to neutrophil engraftment (P = 0.121) and platelet engraftment (P = 0.171) between the two groups. The median duration of neutrophil count under 0.1×10(9)/L and platelet count under 20×10(9)/L in the Bu/Cy group were significantly longer than those in the Bu/Flu group (P = 0.000 and P = 0.047). The incidence of grades II-IV RRT were 68.8% and 25.0% (P = 0.032) in the Bu/Cy and the Bu/Flu groups, respectively. There were no significant differences in the incidence of acute GVHD (P = 0.149), chronic GVHD (P = 0.149), incidence of NRM (P = 0.333), 3-year cumulative relapse rates (P = 0.834), 3-year EFS rate (P = 0.362) and OS rate (P = 0.111) between the two groups.Compared with Bu/Cy, Bu/Flu is a myeloablative condition regimen with milder bone marrow suppression and lower RRT incidence rate in allogeneic HSCT for AML-CR1 patients without compromising the efficacy.

To evaluate the efficacy and safety of bone marrow-derived mesenchymal stem cells (MSC) from a third party donor for secondary poor graft function (PGF) following allogeneic hematopoietic stem cell transplantation(allo-HSCT).Five patients with secondary PGF were treated with MSC at a dose of 1 x 10(6)/kg body weight at a median of 47 days (35 to 61) after secondary PGF. MSC were derived from bone marrow (BM) of HLA-disparate third party donors, cultured in vitro and infused without HSC. If absolute neutrophil cell (ANC) and platelet counts (PLT) did not reach the standardization of1.5 x 10(9)/L and50.0 x 10(9)/L, respectively, within 28-30 days after the first MSC treatment, a second MSC treatment was required.MSC were infused once in one patient and twice in four patients with an interval of 28 to 30 days. All patients obtained ANC and PLT recovery at a median of 34 (25 to 49) days and 47 (26 to 54) days, respectively, without toxic side effects within follow-up periods of median 761 (204-1491) days. Three patients developed Epstein-Barr virus (EBV) reactivation at 42, 48, 108 days after MSC infusion, respectively and two of the three coverted to posttransplant lymphoproliferative disorders (PTLD).MSC from a third party donor are effective to patients with secondary PGF following allo-HSCT, whether it might increase the risk of EBV reactivation and EBV-associated PTLD need further observation.

This study was aimed to establish a method for rapid detecting BK polyomavirus (BKV) and to investigate the feasibility and value used in leukemia patients undergoing hematopoietic stem cell transplantation. Primers were designed according to BKV gene sequence; the quantitative standards for BKV and a real-time fluorescent quantitative PCR for BKV were established. The BKV level in urine samples from 36 patients after hematopoietic stem cell transplantation were detected by established method. The results showed that the standard of reconstructed plasmid and real time fluorescent quantitative PCR method were successfully established, its good specificity, sensitivity and stability were confirmed by experiments. BKV was found in 55.56% of urine samples, and the BKV load in urine was 2.46 × 10(4) - 7.8 × 10(9) copy/ml. It is concluded that the establishment of real-time fluorescent quantitative PCR for BKV detection provides a method for early diagnosis of the patients with hemorrhagic cystitis after hematopoietic stem cell transplantation.

Imatinib mesylate has been commonly used in the treatment of patients with chronic myeloid leukemia (CML). However, a significant number of CML patients treated with imatinib developed thrombocytopenia. Platelet-derived growth factor (PDGF)/platelet-derived growth factor receptor (PDGFR) plays a significant role in the regulation of thrombopoiesis. It is suggested that imatinib may block the PDGF/PDGFR and PI3-K/Akt pathway, then inducing the apoptosis of megakaryocytes and developing thrombocytopenia in these patients. In this review, the potential molecular mechanism of imatinib-induced thrombocytopenia in the treatment of CML patients is discussed, including imatinib and thrombocytopenia, PDGF/PDGFR and thrombopoiesis, potential mechanism of imatinib-induced thrombocytopenia in treatment of patients with CML and so on.

To investigate the effect of granulocyte colony stimulating factor (G-CSF) on myeloid-derived suppressor cells (MDSCs) in the bone marrow and peripheral blood, and explore the relationship between MDSC and graft-versus-host disease (GVHD).Bone marrow, peripheral blood and peripheral blood stem cells were obtained from 12 healthy hemopoietic stem cell donors before and on day 5 after G-CSF mobilization. Flow cytometry was employed to examine the number of MDSC, and the relationship between MDSC number and the incidence of GVHD was analyzed.In normal physiological conditions, MDSC could be detected in the peripheral blood and bone marrow with a cell percentages of (1.35±0.35)% and (2.44±1.11)%, respectively, showing a significantly higher cell percentage in the bone marrow (P=0.015). On the 5th day after G-CSF mobilization, the percentage of MDSCs increased to (4.01±1.82)% in the peripheral blood and to (4.38±2.19)% in the bone marrow, showing no significant difference between them (P=0.083). The mobilization caused a significant increase in the number of MDSCs in the peripheral blood (P=0.047) but not in the bone marrow (P=0.761). The number of MDSCs in the collected samples showed a significant inverse correlation to the incidence of GVHD (P=0.048).MDSCs are present in the peripheral blood and bone marrow of healthy donors, with a greater number in the bone marrow. G-CSF can mobilize the MDSCs from the bone marrow to the peripheral blood to increase number of MDSCs in the peripheral blood, which may contribute to a lowered incidence of GVHD in hematopoietic stem cell transplantation (HSCT).

To evaluate the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the conditioning of different intensities for acute leukemias of ambiguous lineage (ALAL).A total of 38 ALAL patients were treated with two conditioning of different intensities in our hospital from March 2002 to August 2010. The standard conditioning included TBI + Cy or Bu + Cy, intensified conditioning included Fludarabine + Ara-C + TBI + Cy. Cyclosporine A (CsA) and methotrexate (MTX) were administered in patients with human leukocyte antigen-matched sibling donor. And CsA, MTX plus antihuman thymocyte globulin and/or mycophenolate were used in all patients with HLA-mismatched related donor and unrelated donors transplants for graft-versus-host disease (GVHD) prophylaxis. COX regression was used to evaluate the prognostic factors of ALAL.Among 38 ALAL patients, 19 received the standard conditioning while another 19 the intensified conditioning. All patients achieved hematopoietic reconstitution. The 5-year overall survival (OS) and the disease-free survival (DFS) were 35.5% and 25.7% respectively. The 5-year OS rates were 20.2% and 48.1% (P = 0.233) and DFS 6.5% and 43.1% (P = 0.031) in the standard and intensified conditioning groups respectively. The 5-year cumulative relapsing incidence was 58.9% in all patients and 87.6% vs 30.4% in the standard and intensified conditioning groups respectively (P = 0.003). Through a COX regression model for univariate analysis, the intensified conditioning and chronic GVHD were protective factors for DFS (P = 0.001, 0.031).The intensified conditioning in ALAL patients undergoing allo-HSCT may improve the long-term patient survival and decrease the relapse of leukemia. The graft versus leukemic effect has some efficacy in ALAL patients undergoing allo-HSCT.

To analyze the influence of HLA compatibility on the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with chronic myeloid leukemia (CML).This retrospective study involved 121 CML patients including 90 in chronic phase, 8 in accelerated phase and 23 with blast crisis. Of these patients, 85 received related and 36 had unrelated donor allo-HSCT. The conditioning regimens included total body irradiation with cyclophosphamide in 37 patients, and modified BUCY protocol in 84 patients. Cyclosporine A (CsA) and methotrexate (MTX) were used for graft-versus-host disease (GVHD) prophylaxis in patients undergoing HLA-matched sibling donor transplants. CsA, MTX, antihuman thymocyte globulin and mycophenolate were used in all the patients undergoing HLA-mismatched related donor and unrelated donor transplants. The prognostic factors of CML were evaluated using Cox regression and the cumulative overall survival and the disease-free survival were estimated using Kaplan and Meier survival analysis model.The incidence of II-IV acute GVHD was 26.1% in HLA-matched and 53.3% in HLA-mismatched cases (P=0.006), with a 5-year cumulative incidence of chronic GVHD of 47.4% and 49.6%, respectively (P=0.947). The 5-year cumulative incidences of disease relapse was 16.7% in the total patients, with a 5-year cumulative overall survival (OS) of 70.5% and disease-free survival (DFS) of 63.4%. The 5-year OS was 78.2% in HLA-matched cases, as compared with 47.6% in HLA-mismatched cases. Multivariate analysis with Cox regression model identified HLA mismatch, II-IV acute GVHD, and advanced phase as the risk factors affecting the OS.HLA mismatch can significantly increase the incidence of II-IV acute GVHD following allo-HSCT and decrease the long-term survival rate, which is not related to the donor source.

The repair of large segmental bone defects remains a tough problem disturbing surgeons and researchers. Bone tissue engineering brings some new sight in this field. However, it has not been effectively applied in clinics, for the reason that the involved mechanism is not well understood. Thus, we need to know the osteogenesis process of the tissue-engineered bone including distribution, proliferation and interaction among seed cells pre-inoculated in biomaterials as well as the function of surrounding tissues. As a matter of fact, the tissue-engineered bone or the biomaterials are solid and opaque, which makes the study difficult. Here, inspired by the structure of honeycomb and amber, we hypothesize a semisolid decalcification protocol to solve this problem.

To approach the clinical characteristic and risk factors affecting the incidence and outcome of post-transplantation invasive fungal infection (IFI) in patients with malignant hematologic disease and undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT).The incidence and outcome of IFI after transplantation in 193 cases of recipients underwent allo-HSCT in our single center were assessed retrospectively. Some potential influential factors for that of IFI were analyzed by methods of bivariate correlate analysis and binary logistic regression analysis, including the source of donor and stem cell, human leukocyte antigen matched, white blood cell engraftment, the history and states of IFI before transplantation, prophylaxis schema for GVHD, acute and chronic GVHD.The 2-year cumulative incidence of post-transplantation IFI was 34.0% +/- 4.0%. The incidences of breakthrough IFI at primary and secondary prophylaxis were 3.8% and 21.1%, respectively (P = 0.000). 84.2% patients with IFI occurred within half of years after transplantation. In fungus that can be measured, molds and yeasts accounted for 68.1% and 27.7%, respectively. Infective sites in pulmonary or not accounted for were 67.3% and 27.7%, respectively. The total effective rate of IFI was 67.3%, complete response rate was 44.2%. Furthermore, there was no statistically significant difference in the therapeutic effect of antifungal agents between patients with a history of IFI and those without before transplantation. IFI-related mortality was 38.5%. Muti-variate analysis showed acute GVHD was an important factor affecting the incidence and outcome of IFI.Pulmonary mold infection was the most common IFI after allo-HSCT. patients with a history of IFI did not seem to be an contraindication for allo-HSCT. Acute GVHD was a significant risk factor for the incidence and outcome of IFI.

To study the effect of granulocyte colony-stimulating factor (G-CSF) on the proliferation and differentiation of bcr/abl(+)-CD34+ cells.bcr/abl(+)-CD34+ cells were isolated from bone marrow of chronic myelocytic leukemia (CML) patients and were treated with 0, 10, 100, 1000 ng/ml of G-CSF for 48, 96, 144 hs. CD34 cells from normal bone marrow were used as controls. Cell proliferation was determined by trypan blue dye exclusion, cell-cycle and antigen differentiation were determined by flow cytometry and cell morphology was observed under light microscope.The number of bcr/abl(+)-CD34+ cells was increased obviously in all groups. After cultured for 48 and 96 h, the number of bcr/abl(+)-CD34+ cells at G-CSF 10 ng/ml group was significantly higher than that in G-CSF 0 ng/ml group (P0.05) , the number of normal CD34 cells was increased only in the presence of G-CSF. After cultured for 48, 96 and 144 h, the cell number in G-CSF 100 ng/ml group was significantly higher than that in G-CSF 0 ng/ml group (P0.05, P0.01, P0.01, respectively). After cultured for 144 h, the cell percentages in G0/G1 phase for bcr/abl(+)-CD34+ cells in G-CSF 10, 100, 1000 ng/ml groups were significantly less than that in G-CSF 0 ng/ml group (P0. 05), and that for normal CD34 cells in G-CSF 10, 100, 1000 ng/ml groups were significantly less than that of G-CSF 0 ng/ml group after cultured for 48 and 96 h. The expressions of CD34 on bcr/abl(+)-CD34+ cells and normal CD34+ cells were decreased along with the culture duration, accompanied by the expression of CD33 and CD13 increased first and decreased later, which was not correlated with the concentration of G-CSF. Both bcr/abl(+)-CD34+ cells and normal CD34+ cells showed mature morphology along with proliferation and differentiation.G-CSF promotes proliferation of both bcr/abl(+)-CD34+ cells and normal CD34+ cells, but not necessary for the former, and the former differentiates more rapidly than the latter does, but both was independent of G-CSF.

To compare the hemopoietic reconstitution, immune reconstitution, infection, incidence of graft-versus-host disease (GVHD) and clinical outcome between unrelated donor peripheral blood stem cell (PBSC) transplantation and bone marrow (BM) transplantation for leukemias.The clinical results of 21 leukemia patients receiving G-CSF mobilized PBSC graft from unrelated donors were compared with that of 32 patients receiving unrelated BM transplants.Compared with BM grafts, the PBSC graft contained significantly more nucleated cells (P = 0.000), and resulted in a significantly shorter time-to-neutrophil (12.43 +/- 3.67 vs 16.16 + 2.99 days) and platelet engraftment (14.67 +/- 6.19 vs 21.23 +/- 8.25 days), (P = 0.000 and 0.003, respectively). T cell reconstitution between the two groups differed little after transplantation. The incidences of early-stage infection (42.86% vs 53.13%), the probabilities of acute graft-versus-host disease (aGVHD) (61.90% vs 71.88%), the grades III to IV aGVHD (23.81% vs 15.63%), the chronic GVHD (47.06% vs 43.48%) and the probabilities of relapse (6.90% vs 12.50%) between PBSC and BM groups all has no statistical significance (NS). The 2-year disease free survival (DFS) rates of the two groups were (50.14 +/- 12.00) % and (59.81 +/- 8.99)%, respectively also have no NS.G-CSF-mobilized unrelated donor PBSCs engraft more rapidly in the recipients as compared with conventional BM grafts. The T cell reconstitution, the incidence of infection, the incidence and severity of aGVHD and cGVHD, and the 2-year DFS rates between the two groups all have no significant differences.

To compare the effect of unrelated donor bone marrow (BM) transplantation and peripheral blood stem cell (PBSC) transplantation in light of hemopoietic reconstitution, immune reconstitution, infection, incidence of graft-versus-host disease (GVHD) and other complications in patients with leukemia.The clinical outcomes of 16 patients receiving unrelated PBSC graft mobilized by granulocyte colony-stimulating factor (G-CSF) were compared with 30 patients receiving unrelated BM transplantation.Engraftment was achieved in 97.83% of the total patients. Compared with BM transplantation group, PBSC graft contained significantly more nucleated cells (P=0.000), resulting in a significantly shorter time-to-neutrophil (16.21-/+3.09 vs 12.81-/+4.15 days, P=0.003) and platelet engraftment (20.31-/+7.19 vs 15.50-/+6.91 days, P=0.035). T cell reconstitution differed little between the two groups at different time points after transplantation. The incidences of early-stage infection were 37.50% and 50.00% (P=0.644) in the PBSC and BM groups, respectively. In PBSC and BM groups, the incidences of grades I to IV acute GVHD (aGVHD) were 56.25% and 70.00% (P=0.456), 18.75% and 13.79% (P=0.661) for grades III to IV aGVHD, and 30.77% and 36.36% (P=0.413) for chronic GVHD (cGVHD), respectively. The nonrelapse transplant-related mortality (TRM) rates were 18.75% in PBSC group and 33.33% in BM group (P=0.295). The relapse occurred in 18.75% and 6.90% (P=0.226) of the patients in the two groups, respectively, and the 2-year disease-free survival (DFS) rates were 62.19% and 56.23% (P=0.615), respectively.G-CSF-mobilized PBSCs allow more rapid engraftment in unrelated donor recipients in comparison with conventional BM, but T cell reconstitution and the incidence of infection between the two groups differ little, nor are there significant differences in the incidence or severity of aGVHD and cGVHD, nonrelapse TRM or 2-year DFS rates between the two groups.

To construct pMSCV retroviral vector (RV) containing Polo-like kinase 3(plk3) cDNA, a new member of Ser/Thr protein kinase family, and to study the function of plk3 gene by introducing it into human K562 and HL60 cells.plk3 cDNA was sub-cloned into retroviral vector pMSCV-puroR to generate pMSCV-plk3-puroR, pMSCV-puroR being used as the control. RVs were generated by introducing RV vectors into 293T-Amphotropic packaging cells. Titers of RV were measured by NIH3T3 cells with Large-scale-real-time titration method (LaSRT). K562 and HL60 cells were infected with pMSCV-plk3-puroR or pMSCV-puroR viral supernatant by flow-through transduction method, respectively. Following 3-day selection with 3 mg/L puromycin, K562-plk3-puroR, HL60-plk3-puroR, K562-puroR and HL60-puroR cells were obtained. PCR was used to identify the integration of plk3 gene in K562-plk3-puroR and HL60-plk3-puroR cells. Transduction rates were determined by clone-forming ability against puromycin in semi-solid culture medium. Cell growth curve was measured by MTT colorimetry, the cell cycle and apoptotic analysis were detected by flow cytometry, etc.Virus titers generated was (1.31+/-0.65)x10(9)TU/L. The K562 and HL60 transfection efficiency reached 82.3%+/-3.70% and 62.9%+/-4.94%, respectively (n=3). After 3 d puromycin selection, purified K562-plk3-puroR and HL60-plk3-puroR cell were obtained. The proliferation of K562 and HL60 cells was slowed down by plk3 gene transduction. Compared with wild type K562 cells: (1) when cultured in complete DMEM medium, more K562-plk3-puroR cells were observed in G(0)-G(1), 49.7%+/-3.38% vs 43.9%+/-2.34% (P=0.03); (2) when cultured in serum-free DMEM medium for 10 h, fewer K562-plk3-puroR cells were observed in S phase, 43.6%+/-3.74% vs 54.5%+/-1.52% (P=0.02) with lower apoptosis rate, 3.4%+/-0.37% vs 1.3%+/-0.31% (P=0.01).Introduction of plk3 gene into tumor cells with RV vectors can slow down cell proliferation, prevent cells into cell cycle and protect cells from apoptosis in serum-free culture.

To observe the expression of R. gracilis D-amino acid oxidase (DAAO) gene labeled by EGFP in HeLa cells and how it mediates cytotoxicity, DAAO cDNA and EGFP gene were cloned into Eukaryotic expression vector pIRES to construct DAAO gene expression vector pIRES-DAAO. HeLa cells were transfected with pIRES-DAAO in vitro. Expression of EGFP gene in HeLa-D cells was observed under a fluorescent microscope. The efficiency of transfection was analyzed by flow cytometry (FCM). The fluorescent cells were screened by FCM and were named HeLa-D. The activity of HeLa-D cells was detected by MTT colorimetric assay after they were treated with D-Ala at various concentrations. The results indicted that expression of EGFP gene in HeLa-D cells was seen under fluorescent microscope and HeLa-D cells were screened by FCM. Apparently,the prodrug D-Ala killed HeLa-D cells. These results demonstrate that EGFP gene can be regarded as a reporter gene to screen the cells transduced with DAAO quickly, and DAAO/D-Ala suicide gene system may prove helpful in gene therapy of cancer.

To compare large-scale real-time titration method (LaSRT) with standard titration method by flow cytometry (FACS) for determining the titers of green-fluorescence-protein (GFP)-marked recombinant retrovirus.(1) Standard titration method: NIH3T3 cells were inoculated at 2x10(5) /well in 6-well plate, and after cell culture for 12 h, 0.5 ml, 50 microl and 5 microl GFP-marked recombinant retrovirus (n=3) were respectively used to infect the cells, with the final concentration of polybrene being 8 microg/ml. Forty-eight hours later, the cells were treated with trypsin and assayed for the positive rate of GFP by means of FACS. When the positive rate was lower than 10%, the titer was calculated according to the equation: virus titer (TU/ml) =2x10(5)xGFP positive rate/volume of virus stock solution used. (2) LaSRT method: The cells were inoculated at 5,000 cells/well in a 96-well plate, and after cell culture for 12 h, 90 microl/well complete culture medium was used with 8 microg/ml polybrene and 10% newborn bovine serum, 10 microl virus was added into the first well, and ten-fold dilution of the previous virus-containing solution was performed before the virus was added into the next well (8 wells in each group, altogether 3 groups). Forty-eight hours later, inverted fluorescence microscope was used to observe the fluorescence-positive cells in each well. The virus titer was calculated according to the equation: virus titer (TU/ml) =mx10(n+1), where n is the serial number of the reference well, and m the number of positive cells. (3) LaSRT was used to study the influence of freezing/thawing on the titers of recombinant retroviruses.The virus titer obtained with standard method by FACS was (1.54+/-0.38)x10(6) TU/ml, and that of LaSRT was (1.33+/-0.57)x10(6) TU/ml (P0.05). After one cycle of freezing/thawing, the virus titer dropped to (18.1+/-9.9)% (n=7).LaSRT is more rapid and convenient as well as easier to determine the virus titer compared with standard method, and no significant difference is found between the two titration methods.