大鼠胚胎后肾间充质细胞的分离、培养及鉴定  

Isolation,Cultivation and Identification of Metanephric Mesenchymal Cells Derived from Embryonic Rats

作　　者：焦玉清 易著文[1] 何小解[1] 刘喜红[1] 何庆南[1] 党西强[1] 吴小川[1] 莫双红[1] 

机构地区：[1]中南大学湘雅二医院小儿肾脏病研究室湖南省小儿肾脏病临床中心,中国湖南长沙410011

出　　处：《生命科学研究》2008年第1期66-72,共7页Life Science Research

基　　金：国家自然科学基金资助项目(30672251)

摘　　要：分离、培养大鼠胚胎后肾间充质细胞(MMCS),取孕13d大鼠胚胎,分离胚胎肾脏,去除输尿管芽,消化后置37℃、5%CO2、饱和湿度的培养箱中培养.倒置相差显微镜下观察培养细胞,并行HE染色、电镜观察、生长曲线测定;ABC免疫酶染色法检测波形蛋白、角蛋白、nestin、CD133、CD34;直接免疫荧光法检测双花扁豆凝集素(Dolichos Biflorus,DB),并流式细胞术定量检测.MMCS形态为成纤维细胞样,贴壁生长,48～72h达生长高峰;波形蛋白、nestin、CD133表达阳性;角蛋白、CD34、DB表达阴性;MMCS以DB标记后流式细胞检测为单峰.结果表明:培养细胞为后肾间充质细胞,纯度较高并符合干细胞特征.To establish an isolation and culture method for metanephric mesenchymal cells （MMCS）. Rats were anaesthetized on gestation day 13 and metanephric balstemata were separated from ureteric buds by microdissection. Cells were grown in DMEM containing 10% fetal calf serum at 37 ℃ in a 5% CO2 atmosphere. The morphology of MMCS were observed by inverted phase contrast microscope,HE staining, and electronic microscope. The surface antigen phenotypes were identified by immunohistochemistry, and the purification was identified by flow cytometry. A growth curve to be used for studying the characteristics of growing. MMCS grew adherencely as fibroblast-like, positive for vimentin, nestin,and CD 133. Instead, they were negative for keratin,CD34 and Dolichos Biflorus （DB）. The flow cytometry showed a single peak of MMCS. In conclusion,the results suggest that the cultured cells are MMCS,which are coincided with stem cells and with high purity.

关 键 词：后肾 间充质细胞 原代培养 大鼠 

分 类 号：R-332[医药卫生]