不同代次人胎盘间充质干细胞的生物活性比较研究  

Comparative study of biological activities of placenta-derived mesenchymal stem cells at different passages

作　　者：章毅 许惠利 王锋 王哲 李春丽 陈亮 ZHANG Yi XU Hui-li WANG Feng WANG Zhe LI Chun-li CHEN Liang(Shanghai Cord Blood Bank/Shanghai Stem Cell Technology Co., Ltd./China Stem Cell Group Co., Ltd., Shanghai 200051, China)

机构地区：[1]上海市脐带血造血干细胞库/上海市干细胞技术有限公司/中国干细胞集团有限公司,200051

出　　处：《中国医药生物技术》2016年第5期400-406,共7页Chinese Medicinal Biotechnology

基　　金：上海市自然科学基金(16ZR1429200);上海市科委研发平台专项(16DZ2293000);上海市嘉定区工程技术研究中心项目

摘　　要：目的旨在观察不同代次人胎盘间充质干细胞(PMSCs)体外培养的生物学特性和体外诱导分化潜能,为间充质干细胞临床应用安全性及有效性提供实验依据。方法无菌条件下获取人足月胎儿胎盘组织,通过II型胶原酶消化法分离PMSCs,在常规培养条件下培养至第9代,倒置相差显微镜观察P0、P3、P6、P9代次的间充质干细胞细胞形态;MTT活细胞计数法检测细胞生长增殖能力;流式细胞术分析细胞周期及表面标志物的阳性表达率。同时进行成脂、成骨、成软骨诱导分化,诱导14 d后分别进行油红O染色、茜素红S染色、Masson染色鉴定,观察并比较不同代次PMSCs的多向诱导分化能力。结果 PMSCs具有很强的贴壁能力,主要呈纺锤梭形,不同代次的PMSCs有不同的体外增殖能力,以P0、P3代较强;均高表达CD73、CD90、CD105,低表达CD14、CD34、CD45和CD20。细胞周期中G0/G1期细胞所占比例P0为90.95%、P3为88.97%、P6为85.93%、P9为67.89%,诱导分化实验显示,P6、P9代细胞多向诱导分化能力逐渐降低。结论提示在选择胎盘间充质干细胞作为种子细胞时,应注意选择适宜的扩增代数,以便既能保证足够的细胞数目又能保留细胞最佳的分化能力。Objective To observe the biological characteristics and induced differentiation potential of human placenta derived mesenchymal stem cells（PMSCs） at different passages in vitro and to provide experimental evidences for the safety and efficacy in clinical application of PMSCs. Methods The human term placenta were obtained in sterile condition. The PMSCs were isolated by digestion using collagenase II and in adherent cultur in vitro. The PMSCs were cultured into the 9^th generations with the normal cultural condition, and the morphological features of the primary cells, 3^rd, 6^th and 9^th generations of PMSCs were observed. The proliferative ability of PMSCs was detected by MTT assay and the growth curve was drawn. The cell cycle and the positive expression rates of the surface markers of the different generations of PMSCs were detected by flow cytometry（FCM）. Meanwhile, the PMSCs were induced to differentiate into adipocytes, osteoblasts and chondroblasts in vitro, and identified by staining with oil red O, alizarin red, Masson, respectively for 14 d after being induced. The multipotent differentiation potential of PMSCs from the different generation were assessed. Results Placenta derived mesenchymal stem cells were mainly in spindle shaped adherent cells, and PMSCs at different passages had different proliferative capability in vitro, especially the primary cells and 3^rdgenerations of PMSCs with more potent proliferative activities. The PMSCs were strongly expressed CD73, CD90, CD105 and lowly expressed CD14, CD34, CD45 and CD20. The proportion of G0/G1 phase in cell cycle was 90.95% at the primary cells, 88.97% at passage 3, 85.93% at passage 6, 67.89% at passage 9. The multipotent differentiation potential of PMSCs at passages 6 and 9 were decreased. Conclusion The appropriate amplification passage should be chose in order to obtain the cells with best differentiation potential and cell quantity when using placenta derived mesenchymal stem cells as seed cells.

关 键 词：胎盘 间质干细胞 细胞周期 多向诱导分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]