文献类型：期刊文献

中文题名：大鼠肾小球足细胞的原代培养及鉴定

英文题名：Primary culture and identification of rat's glomerular podocytes in vitro

作者：张兆洲[1];戴恩来[1];杨静[1];薛国忠[2];张杰[2]

第一作者：张兆洲

机构：[1]甘肃中医药大学中西医结合学院,甘肃兰州730000;[2]甘肃中医药大学附属医院肾病科,甘肃兰州730020

第一机构：甘肃中医药大学中西医结合学院

年份：2016

卷号：33

期号：6

起止页码：1

中文期刊名：甘肃中医药大学学报

外文期刊名：Journal of Gansu University of Chinese Medicine

基金：国家自然科学基金地区基金项目(81360602)

语种：中文

中文关键词：大鼠;肾小球;足细胞;原代培养;足细胞裂孔膜蛋白;Wilms瘤抑癌因子-1

外文关键词：rat;;glomerulus;;podocyte;;primary culture;;Nephrin;;Wilms' tumor suppressor-1

摘要：目的建立一种操作相对简单、重复性较好的大鼠肾小球足细胞原代培养及鉴定的方法。方法取SD乳鼠,无菌取肾,运用低浓度酶消化法、差异过筛法收集肾小球,将获得的肾小球种植于鼠尾胶原包被的25 cm2细胞培养瓶中,置于37℃、5%CO2恒温恒湿培养箱中孵育。通过倒置显微镜和扫描电镜观察足细胞形态,采用免疫组织化学法检测Wilms瘤抑癌因子-1(WT-1)和足细胞裂孔膜蛋白(Nephrin)的表达以鉴定足细胞。结果种植第4天,可见大部分肾小球已贴壁,第5天见有细胞从贴壁的肾小球中爬出,第9天见约80%的细胞呈铺路石样融合生长。将种植后第9天的细胞消化传代培养,扫描电镜观察可见扁大而平、有树枝状突出的星形细胞。免疫组化结果显示足细胞标志蛋白Nephrin和WT-1表达阳性,表明所培养的细胞为足细胞。结论本实验成功建立了一种操作简便、重复性较好的大鼠肾小球足细胞的分离、培养和鉴定方法,为进一步研究足细胞相关性肾脏疾病奠定了基础;WT-1和Nephrin可作为足细胞鉴定的标记蛋白。
Objective To establish a feasible and repeatable method for primary culture and identification of rat's glomerular podocytes in vitro. Methods Kidneys were isolated from SD rats under sterile condition. The glomeruli were separated by digestion of type Ⅳ collagenase with low concentration as well as variant cells sieve. The glomerular suspension was inoculated into 25 cm2 plastic flask that coated with rat tail collagen and then cultured in constant temperature and humidity chamber with 37 ℃,5% CO2. Cellular morphology was observed through inverted microscope and scanning electron microscope. The expression of Nephrin and Wilms ' tumor suppressor-1( WT-1) were detected through the immunohistochemical method. Results Most glomeruli were adherent on the4 thday,the epithelial cells outgrew from glomeruli on the 5thday and 80% cells took a confluent growth on the 9th day. After consecutive subculturing,cells gradually turned into flat,large and dendric cells. Nephrin and WT-1showed positive expression,which indicated that the cells were podocytes. Conclusion The methods for extraction,culture and identification of rat's glomerular podocytes have been established successfully in the experiment,which lays a solid foundation to study kidney diseases that related to podocyte. Nephrin and WT-1 can be served as proper markers to identify rat's glomerular podocytes.