【摘要】 背景:近年来的研究表明,除已知的人骨髓、外周血和脐带血中存在造血干/祖细胞外,人胎盘组织中也有造血干/祖细胞存在。目前为止,还缺乏对人胎盘组织造血干/祖细胞的增殖分化特性及人胎盘组织淋巴细胞亚群组成和免疫原性等的深入研究。目的:探究人胎盘组织是否含有比脐带血更丰富的造血干/祖细胞,并对其造血祖细胞系增殖分化能力进行检测,同时对人胎盘组织淋巴细胞亚群组成及表型特征进行分析。设计、时间及地点:开放性实验,于2004-01/2006-12在贵州省细胞工程重点实验室完成。材料:经产妇知情同意,无菌采集遵义医学院附属医院产科健康足月分娩新生儿胎盘和脐带血共12份。淋巴细胞亚群检测试剂盒,CD34绝对计数试剂盒(Becton Dickinson公司);CD34磁珠分选试剂盒,FITC标记的CD38单克隆抗体,抗FITC磁珠和MS/LS免疫磁式细胞分选柱(Miltenyi Biotec)。方法:脐带血与RPMI-1640培养基(含体积分数为0.1的胎牛血清)按1∶1的比例混合,采用Ficoll-Histopaque分离液离心30min,吸取界面层细胞,PBS洗涤一次,获得脐带血单个核细胞。采用机械法加0.25g/L胶原酶消化制备胎盘组织单个细胞悬液,之后同脐带血单个核细胞分离步骤分离胎盘单个核细胞。流式细胞仪检测胎盘单个核细胞中CD34+CD38-,CD34+CD38+造血干/祖细胞(HSPCs)和淋巴细胞亚群的组成比例。免疫磁珠分选法分选人胎盘CD34+CD38-、CD34+CD38+造血干/祖细胞,并分别进行粒细胞-单核细胞集落生成单位、红细胞爆裂型集落生成单位、混合集落生成单位系集落形成培养,以评价其造血祖细胞系增殖分化能力。实验全程用脐带血作平行比较分析。主要观察指标:胎盘和脐带血CD34+造血干/祖细胞组成百分率、祖细胞系集落形成能力、淋巴细胞亚群表型及组成特点。结果:①胎盘CD34+造血干/祖细胞百分率是脐带血的8.8倍,差异有显著性意义(P<0.01)。②胎盘中的淋巴细胞总数、T细胞(CD3+CD2+)、B细胞(CD19+)、Th(CD3+CD4+)细胞及Th/Ts比值均明显低于脐带血,而CD8+CD28-T抑制细胞则明显高于脐带血,差异有显著性意义(P<0.01)。③胎盘CD34+CD38+造血干/祖细胞亚群培养形成的粒细胞-单核细胞集落生成单位、红细胞爆裂型集落生成单位、混合集落生成单位集落数明显高于CD34+CD38-造血干/祖细胞亚群(P<0.01);胎盘与脐带血造血干/祖细胞中相同表型细胞亚群形成的各系集落数比较,差异无显著性意义(P>0.05)。结论:人胎盘组织富含CD34+造血干/祖细胞,其CD34+CD38+、CD34+CD38-两个造血干/祖细胞亚群均具有增殖分化为粒细胞-单核细胞集落生成单位、红细胞爆裂型集落生成单位、混合集落生成单位的能力,并且人胎盘组织具有淋巴细胞亚群低比例和抑制性T细胞高比例的特点,使其有望成为造血干/祖细胞移植的新来源。更多还原

【Abstract】 BACKGROUND: As is well known that hematopoietic stem and progenitor cells (HSPCs) contain in bone marrow, peripheral blood, and cord blood. Recent studies found that human placenta tissue (PT) also exists in HSPCs. But so far the property and differentiation capacity of human PT-HSPCs is not yet known. Furthermore the composition of lymphocyte subpopulations and immunogenicity regarded to human PT-HSPCs are also unclear. OBJECTIVE: To verify whether there are more HSPCs in human PT than those in human umbilical cord blood (UCB), to investigate their capacities of proliferation and differentiation, and to analyze the phenotypes of lymphocyte subpopulations in human PT. DESIGN, TIME AND SETTING: Open eXperiments were performed at the Key Laboratory of Cell Engineering of Guizhou Province from January 2004 to December 2006. SETTING: Key Laboratory of Cell Engineering of Guizhou Province, the Affiliated Hospital of Zunyi Medical College. MATERIALS: Twelve human placenta and UCB samples through cesarean delivery were collected aseptically with the informed consents of parturients derived from Maternity Department of the Affiliated Hospital of Zunyi Medical College. The main reagents were detailed as follows: lymphocyte subpopulations analysis reagents Simultest IMK-lymphocyte Kit, CD34 absolute counting reagents Kit (Becton Dickinson); CD34 Multisort Kit, FITC conjugated CD38 monoclonal antibody, anti-FITC microbeads and MS/LS mini MACS segregating columns (Miltenyi Biotec). METHODS: UCB samples were 1:1 diluted with RPMI-1640 containing 0.1 volume fraction of fetal bovine serum and the mononuclear cells (MNCs) were isolated on Ficoll-Histopaque by centrifugation for 30 minutes. The MNCs at the interface were collected and washed with PBS. Single cells suspension liquid of human PT was prepared by mechanical method combined with 0.25 g/L collagenase digestion. After that, the placenta samples underwent the same protocol as used in UCB to isolate MNCs. The percentage of CD34+CD38-, CD34+CD38+ HSPCs and the phenotype of lymphocyte subpopulations derived from human PT-MNCs were analyzed by flow cytometry (FCM). CD34+CD38-, CD34+CD38-cell subsets isolated by magnetic-activated cell sorting (MACS) from human PT were used to carry out colony-forming culture including granulocyte/macrophage colony-forming unit (CFU-GM), burst forming unit-erythroid (BFU-E) and mixed colony-forming unit (CFU-Mix) in order to assess their capacities of hematopoietic progenitor cells’ proliferation and differentiation. In parallel, UCB samples underwent the same protocols for comparison. MAIN OUTCOME MEASURES: Percent compositions of CD34+ HSPCs, hematopoietic progenitors’ lineage colony-forming capacities of CD34+ HSPCs, phenotypes and compositions of lymphocyte subpopulations both in PT and UCB. RESULTS: The percentage of CD34+ cells contained in human PT was 8.8 times higher than that of in UCB (P < 0.01). The total number of lymphocytes, T cells (CD3+CD2+), B cells(CD19+), Th (CD3+CD4+) and Th/Ts ratio were apparently lower in human placenta, while the number of CD8+CD28-T suppressor cells were higher compared to UCB samples (P < 0.01). Among PT, CFU-GM, BFU-E and CFU-Mix frequencies of CD34+CD38-cells subset were much higher than that of CD34+CD38-(P < 0.01). Within the same phenotype of cell subsets, however, the number of each colony-forming unit was similar between PT and UCB (P > 0.05). CONCLUSION: Human PT is richer in CD34+CD38-, CD34+CD38+ HSPCs and both of them have the abilities of proliferating and differentiating into CFU-GM, BFU-E and CFU-Mix. Considering that human PT have a lower lymphocyte subpopulations and higher Ts cells, human PT might be a alternative and suitable source of HSPCs for clinical transplantation.更多还原