胚胎后肾间充质干细胞的分离和生物学特性研究

A study of the isolation and biological properties of metanephric mesenchymal stem cells derived from embryonic rats

目的探讨建立一种简便有效的体外分离纯化大鼠胚胎后肾问充质干细胞（MMSCs）的方法，并研究MMSCs的生物学特性。方法孕14天清洁级SD大鼠，脱颈处死后取其胚胎，分离胚胎肾，剪碎后置于37℃、5％CO2、饱和湿度的培养箱中培养；于倒置显微镜下观察培养细胞形态变化，利用四甲基偶氮唑蓝（MTT）法测定MMSCs的生长曲线，经流式细胞仪检测CD45、CD90及CD133的表达。结果培养细胞呈现梭形或类纤维样，贴壁生长，增殖对数期始于接种后2～3人，5～6天后增殖能力减慢，渐进入平台期。CD45、CD90和CD133表达率分别为1．94％、95．43％和95．31％。结论培养细胞为胚胎后肾问充质十细胞，符合间充质干细胞特性。

Objective To investigate the establishment of an feasible and effective approach to the in vitro isolation of metanephric mesenchymal stem cells （MMSCs） derived from embryonic rats. Methods SD rats of clean class were sacrificed by neck traction on pregnancy day 14 with the embryo removed and metanephron isolated by microdissection. Following metanephronic fragmentation, the cells were cultivated in DMEM containing 10% fetal calf serum in an incubator at 37℃, 5% CO2 and saturated humidity enviroment. The morphological changes of MMSCs were observed by inverted phase contrast microscopy. The growth curves of MMSCs were deten^nined by MTF assay. The expression of CIM5, CD90 and CD133 was identified by flow cytometry. Results The morphology of the cultured MMSCs were presented as shuttle - shaped or fibroblast - like, and wall - adherent in growth. The proliferative logarithm phase occurred two to three days ＇after inoculation and from 5 to 6 days the proliferative ability was retarded and gradually a platform phased followed. The expression rate of CD45, CD90 and CD133 was 1.94%, 95.43% and 95.31% , respectively. Conclusion The cultured cells were identified as metanephrie mesenchymal stem ceils entered, which were consistent with the properties of mesenchymal stem cells with high purity.