摘要
目的:原代培养和鉴定正常人胎肾小球系膜细胞(HMC),探索和总结最佳培养方法。方法:取4月胎龄的引产胎儿肾,分离皮质后,筛网滤过分离肾小球,Ⅳ型胶原酶消化,接种肾小球时分别采用传统方法(第1组)和预先浸湿瓶底并以胎牛血清中和胶原酶替代离心洗涤(第2组)两种方法,观察系膜细胞生长状态。并采用免疫细胞化学检测第3代肾小球系膜细胞的结蛋白(Desmin)、肌动蛋白(Actin)、波形蛋白(Vimentin)、细胞角蛋白(Cytokeratin)、Ⅷ因子(FactorⅧ)等相关抗原的表达,鉴定系膜细胞,并绘制其生长曲线,计算倍增时间。结果:两组肾小球贴壁率分别71%和90%,系膜细胞开始爬出时间为11d和7d,次代倍增时间为3.94d和3.52d,细胞生长状况有统计学差异。第2组传代的系膜细胞1d内贴壁,1周后长满。细胞呈三角、梭形、不规则形半透明,树枝状突起。免疫化学鉴定Desmin、Vimentin和Actin阳性,FactorⅧ和Cytoker-atin阴性。结论:两种肾小球接种方法对系膜细胞培养有不同影响,我们总结改进的培养方法肾小球贴壁率高,细胞存活和生长良好,并确定培养的细胞为人HMC。
Objective：To culture and identify human mesangial cells （HMC） of fetus, investigate and summarize the optimum methods for culturing HMC. Methods：Human fetus aged about 4 months were induced by labor,and then the kidneys were tak- en out and the cortex was separated. Glomeruli were separated using a three- layer microspore filter device and digested with type IV collagenase before inoculation. The glomeruli were divided into two groups in two different ways. The first way was the traditional method. In the second way, the plastic flasks with bottoms were soaked by FCS in advance and the centrifugal clarification was re- placed by the remnants of collagenase with FCS. The cellular markers including Desmin, Actin, Vimentin, Cytokeratin, and Factor vm were tested with immunocytochemistry methods for identification of the HMC. The growth curve was drawn and the doubling time was calculated. Results： There were obvious differences between the two groups, and glomeruli adhesion rates in the two groups were 71% and 90% respectively. HMCs started climbing out at the llth day and the 7th day respectively in the two groups, The sub- generation of HMCs doubled at 3.94 and 3.52 days separately. The second generation of HMCs by the improved method began to adhere in 1 d. About 14 days later, the cells covered the bottom. Observed through a light inverse microscope, HMCs appeared triangle, fusiform, and irregularity in shape and translucence or cytoplasmic projections . The Desmin, Vimentin and Actin were positive in the cultured glomerular mesangial cells by immunocytochemistry staining, while those of Factor Ⅷ and Cytokeratin were negative. Conclusion： Two methods of vaccination have different effects on glomerular mesangial cells. The results suggest that the improved and summarized cell culture method was successful, which increased the cell attachment and survival rate, And the cultured cells from the glomeruli of fetus were identified to be HMCs.
出处
《中国中西医结合肾病杂志》
2009年第4期315-318,I0004,共5页
Chinese Journal of Integrated Traditional and Western Nephrology
基金
甘肃省中青年自然科学基金资助项目(No.YS-011-A23-022)
甘肃省中医药科研基金资助项目(No.GZK-2008-5)
关键词
肾小球
人肾小球系膜细胞
胎肾
免疫细胞化学
原代培养
Glomerulus Human mesangial cells （HMC） Human fetus kidney Immunocytochemistry Primary cellculture
