骨髓间质干细胞的分离培养与鉴定  

Isolation, incubation and identification of bone marrow-derived mesenchymal stem cells

作　　者：蒋文慧[1] 马爱群[1] 董安平[1] 冯敏[1] 郑小璞[1] 王亭忠[1] 耿涛[1] 许正云[1] 

机构地区：[1]西安交通大学第一医院心血管内科,教育部环境与疾病相关基因重点实验室,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2005年第3期224-227,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：西安交通大学重点培植项目(XY10082008)

摘　　要：目的建立兔骨髓间质干细胞(MSCs)的体外分离纯化、扩增和鉴定的方法,为MSCs的进一步诱导分化和应用奠定基础。方法抽取兔胫骨骨髓,密度梯度离心,得到骨髓单个核细胞,接种后形成单层贴壁细胞,经胰蛋白酶消化后传代培养扩增,倒置显微镜下观察原代和传代细胞的生长特性。通过免疫荧光组织化学、流式细胞分析和透射电镜鉴定细胞。结果分离培养的骨髓MSCs贴壁、呈梭形,原代细胞呈集落生长,7～10d达到融合,传代后增殖速度加快;骨髓MSCs表达CD90,反复传代后表达效率增高,不表达CD34;透射电镜可见细胞表面有大量的绒毛突起,胞浆中有丰富的粗面内质网和线粒体,核大,形态不规则。结论密度梯度离心结合贴壁法,以及消化传代能有效分离纯化和扩增骨髓MSCs,分离培养的细胞具有骨髓MSCs的基本表型和特性。Objective To establish a method for the isolation and cultivation of rabbit mesenchymal stem cells (MSCs) from bone marrow and to study their phenotypical properties to offer an experimental foundation for the further differentiation and actual application. Methods MSCs were isolated from bone marrow and purified by density centrifuge and adhered to the cultural plastic in vitro. Then, the cells were expanded by subculture successively. The proliferation and growth characteristics were observed with phase contrast microscope in primary and passage culture. MSCs quality was evaluated with immunocytochemistry, FAC Scan flow cytometer, and transmission electronmicroscope (TEM). Results MSCs had spindle shape and a typical morphological fibroblast. The primary cells were confluent in single layer after being plated for 7～10 days. The cells were noted to have a great expansive potential after subculture, and they uniformly expressed positive for CD90 and negative for CD34. Large number of villus on surface and rough surfaced endoplasmic reticuli and mitochondria in endoplasm were observed in cells through TEM. Conclusion MSCs can be isolated and purified from postnatal bone marrow through density centrifuge, adherence ability and subculture. The cultured cells have basic phenotypical properties and characteristics in MSCs.

关 键 词：骨髓间充质干细胞 分离培养 鉴定 兔 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]