Retrovirus (RV) is the most widely used vector for gene transfer, but its efficiency is very low for human cells. A Flow-through Transduction System made in General Hospital of PLA was used to deliver interleukin-12 RV vector into human leukemia cell line K562. Compared with static procedure, the efficiency could be significantly improved,and detected by clony forming units (CFU) for NeoR gene in semisolid culture medium with G418 ( 83.1%?3.9% vs 9.2%?2.3%, P0.05), while statically transducted cells without G418 selection could express very low IL-12 (0.1?0.024ng/ml,P

Objective To study the therapeutic efficacy of HSV-TK/GCV in acute graft versus host disease (GVHD) produced by infusion of H-2 haplotype matched CD34 + cells in combination with TK +T lymphocytes transplantation. Methods CD34 + cells were separated from bone marrow of male donor mice, and T lymphocytes were acquired from the spleen and transfected with retroviral vectors carrying tk gene. Forty-eight female F1 recipient mice were divided into six groups. Group 1 received 1?10 5 CD34 + cells, groups 2 and 3 received 1?10 5 CD34 + cells and 1?10 7 T lymphocytes, groups 4～6 received 1?10 5 CD34 + cells and 1?10 7 TK +T lymphocytes. GCV was administered to group 5 and group 6 in the dosage of 50mg/(kg?d) for 7 days by intraperitoneal injection on day 0 and 7 posttransplant, respectively. Results All the recipient mice in group 1 survived more than 30 days and no acute GVHD symptoms emerged. Acute GVHD symptoms appeared in the recipient mice in groups 2～6 showing typical pathological manifestations. The recipient mice in groups 2～4 died of acute GVHD 3 weeks posttransplant. The survival time of recipient mice in groups 5 and 6 was 26.6?4.8 days and 40.5?8.4 days, respectively, which was significantly longer than that of groups 2～4 (P

Retrovirus (RV) is the most widely used vector for gene transfer, but its efficiency is very low for human cells. A Flow-through Transduction System made in General Hospital of PLA was used to deliver interleukin-12 RV vector into human leukemia cell line K562. Compared with static procedure, the efficiency could be significantly improved,and detected by clony forming units (CFU) for NeoR gene in semisolid culture medium with G418 (83.1%±3.9% vs 9.2%±2.3%, P＜0.01). No significant difference was found in the levels of IL-12 p70 protein expression detected by ELISA, by flow-through transducted cells without G418 selection and by statically-transducted cells with G418 selection (11.3±7.5ng/ml vs 12.0±6.4ng/ml, P>0.05), while statically transducted cells without G418 selection could express very low IL-12 (0.1±0.024ng/ml，P＜0.01)。The Flow-through Transduction System is efficient and easy for use, it can be widely used in research and clinic of gene therapy.

Objective Clinical research was made to observe the relationship between idiopathic thrombocytopenic purpura(ITP)and Helicobacter pylori(ITP)and to evaluate the effect of HP eradication in the treatment of chronic ITP.Methods ~(14)C urea breath test were made in 17 patients with chronic idiopathic thrombocytopenic purpura,including 6 males and 11 females,aging from 15-48,with course of disease from 4 months to 8 years.Omeprazole,clarithromycin and amoxicillin were used for Helicobacter pylori eradication,and the effect was evaluated by platelet count.Results Thirteen out of 17 patients were HP positive,and eleven turned negative after HP eradication,eight of whom were observed platelet recovery.Conclusion Platelet count can be raised in some patients with chronic ITP after eradication of Helicobacter pylori.

Objective To morphologically evaluate the bone marrow (BM) smear specimens collected from the patients with chronic myelogenous leukaemia (CML) in accelerated phase and blastic phase receiving a short term Gleevec therapy, and to determine its clinical significance in evaluating the changes in disease condition to guide the treatment. Methods Sequential BM smear specimens of 16 Ph positive CML patients, including 9 in accelerated-phase and 7 in blastic-phase, were examined before and 3,6 and 9 weeks after Gleevec treatment (0.4/d or 0.6g/d, PO) with routine method. Periodic acid-Schiff reagent (PAS) staining was performed for a proper identification of abnormal erythroid precursor cells. Results The treatment rapidly caused following conspicuous BM changes in the process: both cellular proliferation and neutrophil granulopoiesis decreased significantly, while the accumulation of erythroid precursor cells increased obviously, and megakaryocytes decreased markedly at 3rd week. A few patients showed no such changes, but an accumulations of erythroid precursor cells, leading to misdiagnosis in some one third of the patients. In 21 percent of patients, in whom no erythroid cells were found, were classified into accelerated phase and blast phase based on both WHO classification system and FAB system. In fact the increase in red system was a bone marrow response to anemia caused by Gleevec therapy. If a condition of severe cellular proliferation of BM with a lower WBC and platelets appeared, most patients could not tolerate the therapy, then it should be ceased. Conclusion Initial treatment with Gleevec therapy exerts pronounced changes in BM morphology, which can be used as a simple and effective method to evaluate the outcome of the patients.

Objective To explore the difference of immunosuppresive effect between the expanded Stro-1+ and Stro-1-subgroups of human mesenchymal stem cells in vitro. Methods Mononuclear cells (MNCs) were isolated from bone marrow (BM) samples and seeded in a T-75 cm2 tissue culture flask contained with Dexter medium. When 50% confluence was obtained, adherent cells were collected and incubated with anti-stro-1 antibody, and the Stro-1+ and Stro-1-MSCs were further seeded for expansion. The total culture time (median) was 15 days. Cells were then analyzed by flow cytometry. One-way mix lymphocyte reaction (MLR) (1?105 responding cells and an equal number of stimulating cells/well were co-cultured in 96-well plates) and nonspecific mitogenic stimuli phytohemagglutinin (PHA) plus interleukin-2 (IL-2) induced lymphocytes proliferation (PBL 1?105/well were mixed with PHA 10?g/ml and IL-2 500U/ml in 96-well culture plates) were established in vitro. 1?103-3?104 irradiated Stro-1+ MSCs and Stro-1-MSCs were added into the two systems at the beginning of reaction. Immunosuppressive actions of both Stro-1+ or Stro-1-MSCs were compared. Results Adherent cells contained a median of 9% (range 5%-26%) Stro-1+ cells, which expressed higher immunophenotype of MSCs. In both reaction systems, suppressive actions occurred in a dose-dependent fashion when whatever Stro-1+ MSC or Stro-1-MSC were added. However, that the addition of 1?103 Stro-1-cells enhanced rather than inhibited the lymphocyte proliferation in one-way MLR. In the presence of various concentrations of MSCs, Stro-1+ MSCs always showed a significantly increased inhibitory effects in comparison to Stro-1-MSCs (P

35), while the expression level of TGF-?1 in Stro-1-MSC was significant higher than that detected in Stro-1+MSC (2-??Ct=0.39, P

C57BL/6 synergistical mice were divided into 4 groups (10 mice each). The first group was negative control without any interference. Each mouse in the other 3 groups was subcutaneously inoculated with 1 10 6 wide type (wt) EL4 tumor cells. Then each group was treated either with interleukin 12 (mIL 12) in vivo vaccine or NeoR control vaccine or PBS(positive control group) on day 1, 7, 14, 21, on the same place where wt EL4 tumor was inoculated. mIL 12 in vivo vaccine and NeoR control vaccine were package cells which can produce retrovirus (RV) with mIL 12 or NeoR gene, the vaccine was 60 Co irradiated and injected (1?10 7 cells/mouse). All mice in positive group and NeoR control group died of tumors in a month, while 5/10 mice in mIL 12 in vivo vaccine groups survived without tumors for more than 60 days. The 5 survived mice were rechallenged with 5?10 5 wt EL4 cells, 3/5 mice even survived without tumors for another 60 days. Among the mice with tumors in vivo vaccine group mice, compared with the controls, the development of tumors was delayed ( P

Objective To establish a method for detecting chimerism after allogeneic stem cell transplantation using microsatellite polymorphism. Methods DNA was extracted from bone marrow or peripheral blood of 10 recipients and their donors, DNA fragments including 5 microsatellite loci were amplified by PCR, the polymorphism of PCR products was analyzed by PAGE and sliver staining, and the quantitative analysis of chimerism was performed using image analysis software. Results [WTBZ]The five selected STR loci had high polymorphism, and were suitable for detecting chimerism. The sensitivity of sliver staining was 90%. Conclusion Microsatellite polymorphism analysis based on PCR is a sensitive and accurate method to detecting chimerism after allogeneic stem cell transplantation, but the sensitivity of sliver staining was not so good,so more sensitive methods should be used.

Objective To explore the application of particle-gun technique in transfecting dendritic cells(DCs),and investigate the expression of DNA vaccine particle IgHV1-GM-CSF in DCs induced by particle-gun.Methods Monocytes were isolated from donor peripheral blood collected by Ficoll density gradient method and adherent culture,and then differentiated into immature DCs(imDCs) by rhGM-CSF and rhlL-4 induction.The plasmids for transfection were IgHV1-GM-CSF/pcDNA3.0 and pEGFP,which were prepared with endofree plasmid purification kit.The DCs were transfected with plasmid pEGFP by particle-gun at different transfection conditions,the green fluorescence positive cell and the total cell numbers were counted respectively under fluorescence microscope,and the transfection efficiency was calculated;the viable cell count was performed after trypan blue staining;the transfection efficiencies of particle-gun,liposome-mediated transfection and electroporation method were compared.The plasmids IgHV1-GM-CSF/pcDNA3.0 and pEGFP were cotransfected into imDCs by particle-gun under the optimum conditions,and then DNA was extracted,the expression of IgHV1-GM-CSF was determined by RT-PCR,and of GM-CSF by ELISA.Results The optimum conditions of particle-gun transfection in imDCs were: gas pressure at 120psi,PVP concentration in 0.02mg/ml,microcarrier loading in 0.5mg gold/shot,and DNA loading ratio in 1.5?g plasmid/mg gold.The transfection efficiencies of particle-gun and electroporation were 10.5%?2.4%(n=3) and 45.2%?5.6%(n=3) respectively,while that of liposome-mediated transfection was very low,and significant difference existed among the three methods(P