BMSCs分离培养、纯化及鉴定.doc

BMSCs分离培养、纯化及鉴定 　 作者：马民 吕志刚 杜以宽 张桂娟 马义 【摘要】 目的 建立一种持续、稳定的可多向分化的骨髓间充质干细胞（BMSCs）体外分离培养体系。方法 运用密度梯度离心法从1月龄新西兰兔骨髓中分离培养BMSCs，利用相差显微镜观察其形态及生长情况，扫描电镜观察其细胞结构，运用流式细胞术分析分离细胞群所处细胞周期和细胞活力，用MTT比色法绘制细胞生长曲线，并用特定诱导液将分离的BMSCs向成骨细胞和成脂肪细胞定向诱导分化，利用ALP和油红O进行染色鉴定。结果 所分离的BMSCs细胞在形态学观察与生长动力学上均符合BMSCs特征，分离培养的BMSCs细胞在第3天进入对数生长期，第10天进入平台期；在成骨、成脂肪的诱导培养条件下，分别出现成骨、成脂肪表型特征，可进一步定向分化，结论 所收获的细胞具有BMSCs的特异性。 【关键词】 骨髓间充质干细胞；分离；成骨分化；成脂分化 【Abstract】 Objective To establish a sustained and stable bone marrowderived mesenchymal stem cells (BMSCs) isolation and culture system which could be pluripotential in vitro. Methods BMSCs were isolated and cultured from the 1monthold New Zealand white rabbits through densitygradient centrifugation. Growth and ultromicrostructure of the BMSCs were observed by lightmicroscope and scan electronmicroscope. The cell activities and cell cycle were observed by flow cytometry. The grow curve of the BMSCs was described through MTT assay. BMSCs were induced and differentiate into osteoblasts and adipocytes by specific induction culture medium. Then the cells were stained with alkaline phosphatase (ALP) and Oil red O , and analyzed the result of all staining. Results The isolated BMSCs cells were in line with the characteristics of BMSCs in morphology and growth kinetics, and the isolated BMSCs cells would be into the logarithmic growth phase in the third day and into the platform phase in the tenth day. Conclusions In the osteogenic and adipogenicinduced culture conditions, BMSCs have appeared the phenotypic characteristics of osteogenic and adipogenic respectively, and could be further directed differentiation. These indicated the harvested cells possessed BMSCs specificity. 　　【Key words】 Bone marrowderived mesenchymal stem cells(BMSCs); Isolation; Osteogenic differentiation; Adipogenic differentiation 　　软骨组织工程技术〔自体软骨细胞的移植、生长因子对软骨细胞的作用、骨髓间充质干细胞（又称骨髓基质干细胞，BMSCs）在软骨组织工程中的作用、转基因技术〕是当今的研究热点〔1，2〕。特别是自体BMSCs，因其低免疫原性、无限扩增性及多向分化潜能等诸多优点而引起广泛关注，成为软骨组织工程种子细胞的首选来源。本实验选择合适的BMS