成人肾脏足细胞的原代培养和鉴定

Primary culture and identification of human glomerular podocytes

目的建立一种重复性好、操作简便的成人肾脏足细胞原代培养方法。方法取肾脏肿瘤手术切除后远离患病部位的正常肾脏组织，分离出肾皮质，剪碎研磨并通过差异过筛法分别通过80目（孔径220μm）、40目（450μm）、120目（125μm）细胞筛网，收集120目筛网上肾小球利用植块法进行接种，将接种培养面向上放置，4h后添加培养液并将培养瓶翻转过来正常放置培养箱内孵育。采用形态学观察和细胞间接免疫荧光染色法，对去氧肾上腺素、第八因子（F8）蛋白、波形蛋白、角蛋白和肾母细胞瘤蛋白（WT-1）进行鉴定；并用流式细胞仪分析足细胞纯度。结果接种3d后可见绝大部分肾小球贴壁；5d后几乎全部肾小球贴壁，少许肾小球周围有细胞爬出，体积中等，呈多边形，无突起伸出；7～10d可见所有肾小球周围大量多边形细胞爬出，细胞迅速生长至融合状态，呈铺路石样外观，符合去分化状态足细胞特征；此时胰蛋白酶差异消化去除成纤维细胞传代培养。消化传代后的足细胞胞体、胞核逐渐增大，自细胞体伸出明显树枝状突起，常见双核，符合分化状态足细胞特征。免疫荧光染色发现传代7d后细胞表达足细胞特异性蛋白WT-1、去氧肾上腺素，不表达F8蛋白、波形蛋白、角蛋白，排除内皮细胞、系膜细胞和壁层上皮细胞污染。流式细胞仪分析足细胞纯度为98．3％。结论应用差异过筛法分离人肾小球，并结合植块法进行接种可促进肾小球贴壁，简便、高效地培养出原代足细胞。

Objective To establish a repeatable and feasible method for culturing human glomerular podocytes in vitro. Methods The renal epithelial tissues were obtained from the tumor-free pole of adults＇ kidneys after kidney tumor resection. Decapsulated human cortical slices were pressed through a series of stainless steel sieves （ sieving method） with increasing pore sizes of 220 to 450 μm in a sterile, clean environment; as a final step, the glomeruli were collected on a 125 μm sieve and then cultured in 25 cm2 flasks of which bottoms were soaked by RPMI 1640 medium with 10% fetal bovine serum （FBS） in advance. Made the flasks upside down （ explant method） ; 4 hours later, added medium and normally placed them. Cells were kept at 37 ℃ in a humid atmosphere under 5% CO〉 Glomerular podocytes cellular markers as nephrin, Wilms tumor protein （ WT-1 ） , factor Ⅷ, vimentin and cytokeratin were analyzed by morphology and indirect immunofluorescence staining method, respectively. The purity of podocytes were determine by flow cytometry technology. Results Most of the glomeruli adhered to the wall of culture dish on the 3 rd day. Almost all glomeruli adhered on the 5 th day with a few climbed out polygonal cells which lost both primary and foot processes and appeared as cobblestones. A larger number of cobblestone-like cells which exhibited strong proliferative activity outgrowth from nearly every glomerular around from 7 th to 10 th day. Then the cells were digested by trypsin differences digestion method to remove fibroblast and subcuitured. In subculturing, podocytes differentiate into other phenotypes which were large, branched, binucleated and exhibited no proliferative activity. It was observed by immunofluorescent staining that the cells were in line with characteristics of podocytes and expressed WT-1 and nephrin, but not the factor Ⅷ, vimentin and cytokeratin with no pollution of endothelial cells, mesangial cells and parietal epithelial ceils. Furthermore, purity of podocytes was 98. 3% checked by flow cytometry analysis. Conclusion It is simple and highly effective to culture primary cultured glomerular podocytes by combined sieving and explant method.