长期培养人脐带间充质干细胞分化能力改变的研究  

Study on Differentiation Potency Changes of Long-term Cultured Human Umbilical Cord MSCs

作　　者：柳菁[1] 宇丽[1] 许超[2] 罗二梅[1] 

机构地区：[1]暨南大学医学院生物化学教研室广州510632 [2]暨南大学医学院中心实验室广州510632

出　　处：《中国生物工程杂志》2012年第2期8-16,共9页China Biotechnology

基　　金：广东省自然科学基金资助项目(9151008901000050)

摘　　要：目的:分析体外传代培养至23代,人脐带间充质干细胞(human umbilical cord-MSCs,hUC-MSCs)多向分化能力的改变,以探索hUC-MSCs体外诱导分化的最佳时间窗。方法:采用胶原酶消化法提取hUC-MSCs,并用胰酶消化传代;收集不同代细胞,用流式细胞术检测细胞表面抗原及细胞周期;MTT法检测不同代细胞的增殖活性;取不同代细胞进行成骨、成软骨及成脂肪诱导鉴定;并利用实时定量PCR分别检测OCT-4、SOX-2、Nanog的mRNA表达水平。结果:用胶原酶消化法可获得形态均一,可稳定传代23代以上的hUC-MSCs;体外传代培养至23代,细胞表面标志物表达率无明显改变;细胞生长曲线形态相似;细胞周期亦无明显差异,(73.04±1.15)%的细胞处于G0/G1期;细胞均可被诱导成骨细胞、软骨细胞和脂肪细胞;不同代细胞OCT-4、SOX-2、Nanog的mRNA表达无显著差异。结论:体外培养至23代,随着传代次数的增加,hUC-MSCs的多向分化能力并未受影响。Objective ： To investigate the ability of muhilineage differentiation of human umbilical cord-MSCs by subculturing to 23 passages. Methods： Mesenchymal stem cells were isolated from umbilical cord using collagenase 11 digestion and were digested by trypsin for continuous passage culture. A series of different generation cultured-cells were collected for following experiment. The cells surface antigens and cell cycle were measured by flow cytometry; The cell growth curve was analyzed by MTT assay; Adipogenic, osteogenic and chondrogenetic ability were evaluated by specially inducing culture; OCT-4 ,SOX-2 and Nanog mRNA expressionlevel were assayed by real-time fluorescence quantitative PCR. Result： MSCs can be obtained by conagenase digestion method. They had uniform shape, and can be stably passaged over 23 generations. By subcuhuring to 23 passages in vitro, the expression rate of surface marker in cell was not significant changed; the shape of cell growth curves were similarity; there was also no obvious difference in cell cycle, （73.04 ± 1.15 ）% of the cell were G0/G1 phase; cells can be induced into osteoblasts, chondrocytes and adipocytes; OCT-4, SOX-2 and Nanog mRNA expression level were no obvious difference. Conclusion ： Subcuhuring to 23 generation in vitro, the ability of mutilineage differentiation of hUC-MSCs was not imCluenced with increase in numbers of subculture.

关 键 词：人脐带间充质干细胞长期培养 多向分化基因 

分 类 号：Q254[生物学—细胞生物学]