摘要
目的研究脐带血来源的间充质干细胞(mesenchymal stem cells,MSCs)体外分离、纯化及培养的条件,以建立稳定的体外培养体系,满足实验和临床的需要;探讨经尾静脉移植脐血间充质干细胞对心肌梗死大鼠心功能的影响。方法无菌条件下采集健康育龄产妇正常分娩胎儿的脐带血42份,脐血随机分成3组:FBS(胎牛血清)未包被组、FBS包被组和mesencult培养基组。FBS未包被组和FBS包被组均使用含10%FBS的LG-DMEM培养基、mesencult培养基组使用专用来培养干细胞的mesencult培养基,与其添加物配合使用。3组均用Ficoll淋巴细胞分离液,密度梯度法分离脐血单个核细胞(MNC),将单个核细胞按密度1×108/ml接种于T25培养瓶中,置37℃饱和湿度含5%CO2孵育箱培养,72 h后全量换液,去除未贴壁细胞,以后均7 d换液1次。待细胞长到80%铺满瓶底时结束原代培养。按1∶1进行消化传代。观察不同培养条件对脐血MSCs生长的影响。取P2代细胞用流式细胞仪检测细胞表面CD 29、CD 34、CD45、CD 105标志。将36只SD大鼠随机分成MSCs移植组、假手术组和心肌梗死植组各12只,结扎左冠状动脉前降支制备大鼠心肌梗死模型。心肌梗死后1周,经尾静脉注射脐血MSCs,4周后测定大鼠血流动力学。结果 42份脐血中单个核细胞原代培养,其中仅8份传代培养成功,而其中mesencult条件培养基有5份培养成功,明显高于FBS包被组(3份)和FBS未包被组(0份)。流式细胞仪检测第2代的脐血MSCs结果显示,P2代MSCs不表达或极弱表达CD 34,CD45造血细胞标志,稳定地高表达CD 29、CD 105间充质细胞相关的表面抗原标记。这与骨髓MSCs的表面抗原标志相一致。移植后4周,经血流动力学检测,与心梗组比较,MSCs移植组左室心功能明显改善(P<0.05)。结论将脐血单个核细胞以高密度(1×108cells/ml)接种在mesencult培养基中可以在体外成功培养出较纯化的脐血MSCs,其培养成功率较高。脐血MSCs的免疫表型符合间充质干细胞特征。脐血MSCs移植能促进心梗大鼠心功能恢复。
Objective To investigate the isolation,purification and culture of human umbilical cord blood(HUCB)mesenchymal stem cells(MSCs)in vitro,and built stable culture system to satisfy experimental and clinical needs.then the human umbilical cord blood mesenchymal stem cells was transplanted into the rats of acute myocardial infarction(AMI)to observe the effect for heart function. Methods Human umbilical cord blood samples(n=42)were collected from healthy mothers.ALL samples was divided into 3 groups,groups 1 not coated with fetus bovine serum(FBS),groups 2 coated with FBS,groups 3 culture medium consisted of Mesencult(a kind of medium special for stem cell cultured),3 groups was isolated by lymphocyte separation medium using the Density-Gradient Technique.and inoculated with a concentration of 1×108 cells/ml in a 25T culture flask.The cells were incubated in a homeothermia culture chamber at 37℃,5%CO2,in a fully humidied atmosphere.After culture for three days the medium was replaced and non-adherent cells were washed out with the all medium changed.Then the all medium changed every six days.Once adhered cells reached approximately 80% confluence,cells were harvested and re-plated at 1:1 under the same culture conditions.detected the second generation of MSCs' immunophenotypes(CD 29,CD 44,CD 45,CD 105)with flow cytometry(FCM).36 SD rats were randomly divided into three groups:MSCs transplantation group,sham group and AMI group,28 days after transplantation.all the rats were performed invasive left ventricular(LV)hemodynamic measurement. Results 42 human umbilical cord blood samples that carried out original MNCs culture,only 8 succeeded in subculture.The succeed rate of culture Mesencult group was more high non-coated with FBS group and coated with FBS group,flow cytometer detected The second generation of HUCB-derived MSCs' immunophenotypes showed the 2th generation of HUCB-derived MSCs did not express CD 34,CD 45 hematopoietic cell marks or weakly expressed,but highly expressed CD 29,CD 105 mesenchymal cell's relative surface antigen marks.after 4 weeks,The invasive left ventricular(LV)hemodynamic measurement result revealed left heart functions in MSC transplantation group was more obviously improved compared with those of myocardial infarction group and sham group. Conclusion It is possible to obtain MSCs from HUCB with remarkable MSCs-specific immunophenotype.In the condition of a culture medium with mesencult,a culture flask and an account of more than 1×108 MNC,MSCs in HUCB can be cultivated successfully in vitro with high achievement ratio and purity.Transplantation of HU-MSCs improved heart function on rat heart of AMI.
出处
《血栓与止血学》
2010年第6期244-249,共6页
Chinese Journal of Thrombosis and Hemostasis
基金
广东省科技计划资助课题(编号:2009B030801358)