大鼠肾小球足细胞原代培养及鉴定方法的研究-第三军医大学学报.docVIP

大鼠足细胞原代培养及鉴定方法的研究 贾苗，张益民，赖德源* （中山大学孙逸仙纪念医院肾内科，广州 510120） 提要： 目的 建立一种重复性好、操作简便的大鼠足细胞培养方法。方法 取重量200-220g的健康雌性SD大鼠，无菌取肾后通过差异过筛法分别通过80目、150目及200目细胞筛网，收集200目筛网上肾小球进行接种，利用植块法将接种培养面向上放置，4.5h后将培养瓶翻转过来正常放置于37℃、5%C02的恒温恒湿培养箱进行孵育。采用形态学观察和细胞间接免疫荧光技术，对足细胞进行鉴定，并用流式细胞仪进行足细胞纯度分析。结果 5d后可见绝大部分肾小球贴壁并有少许肾小球周围有细胞爬出，7-8d可见所有肾小球周围有大量细胞爬出，胰酶消化后传代培养。形态学及特异性抗体WT-1, nephrin鉴定为足细胞。流式细胞仪分析细胞纯度99%左右。结论 三层筛网法分离大鼠肾小球，结合植块法促进肾小球贴壁，可以简单、高效地培养出原代足细胞，且具备足细胞生物学特性，且在操作简单的情况下细胞产量、纯度与国外文献报道的相当。关键词： 足细胞；细胞原代培养；大鼠 中图法分类号： R322.61； R329.24 文献标识码：A Primary cell culture and identification of rat glomerular podocytes Jia Miao , Zhang Yi-min, Lai De-yuan* (Department of Nephrology, Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University, Guangzhou 510120,China) Abstract： Objective To establish a easy-replicated method for primary culture of rat glomerular podocytes. Methods Kidneys were isolated from the normal SD rats weighted for 200-220g under aseptic condition. With differential sieving and explant culture methods , glomeruli were collected on the 200-mesh screen after being pushed through 80-mesh and 150-mesh screens, and then explanted on the culture flasks in the incubator. Podocytes were digested and plated on the 24-well culture dish and characterized by the morphology method and the immunohistochemical method using indirect immunofluorescence staining as a label for WT-1 and nephrin. Flow cytometry technology was used to determine the purity of podocytes. Results Most glomeruli adhered to the culture dishes on the fifth day, podocytes were subcultured after 7 to 8 days. The staining of WT-1 and nephrin was positive. The purity of podocytes was about 99%. Conclusion The differential sieving and explant culture methods are high-effective and convenient for basic research, most importantly, the product of podocytes is comparable to the isolation method with magnetic beads. Key words： Glomerular podocytes; Primary cell culture; Rat近年来随着对肾小球疾病发病机制的深入研究发现足细胞损伤是导致其持续进展的关键细胞[1]，如微小病