大鼠足细胞的原代培养及其Nephrin的特异表达  

Primary culture of rat glomerular podocytes and its specific expression of Nephrin

作　　者：顾杰[1] 张春江[2] 贾林[2] 杨晓萍[2] 

机构地区：[1]石河子大学医学院,新疆石河子832003 [2]石河子大学医学院第一附属医院肾内科,新疆石河子832008

出　　处：《广东医学》2017年第6期825-829,共5页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81160090)

摘　　要：目的建立简便的大鼠足细胞原代培养体系,检测其去氧肾上腺素(Nephrin)mRNA及蛋白的表达水平。方法无菌取肾,分离肾皮质,胶原酶消化法分离肾小球,培养9~10 d,胰蛋白酶差异消化法传代,过筛去除肾小球继续培养5~7 d:形态学观察和间接免疫荧光法进行足细胞鉴定;流式细胞仪分析足细胞纯度;实时定量PCR(qRT-PCR)、间接免疫荧光法和Western blot法比较前三代足细胞中Nephrin mRNA及蛋白的表达水平。结果接种5 d后大部分肾小球贴壁,肾小球周围有细胞爬出,其最外缘有树枝状细胞分布,形态学及特异性抗体Nephrin、WT-1鉴定为足细胞;流式细胞仪分析足细胞纯度为94.7%;间接免疫荧光和qRT-PCR均显示:与第二、三代足细胞相比,第一代足细胞中Nephrin mRNA和蛋白表达均较高(均P<0.05);Western blot示体外培养大鼠足细胞表达两种分子量的Nephrin,且第一代足细胞Nephrin灰度值高于第二、三代足细胞(P<0.05)。结论酶消化法结合差异过筛法接种可促进肾小球贴壁,有利于培养纯度高的足细胞;大鼠足细胞体外培养条件下能特异性表达Nephrin结构及功能蛋白。Objective To establish a repeatable and simple method for culturing rat glomerular podocytes in vitro and to study the significant expression of podocyte marker, Nephrin. Methods Sterile kidneys were extracted from male SD rats. The glumeruli were ground and separated by enzymatic digesting and sieving （100 mesh, 150 mesh, and 200 mesh） , and subsequently exposed in culture medium at a constant temperature incubator for 9 days. The podocytes grow- ing from glumeruli were digested and passaged to different container according to the corresponding experimental demands. The distribution of Nephrin and WT - 1 were detected by cell morphological observation and indirect immunofluorescence staining method. The purity of podocytes were determine by flow cytometry technology. The specific expression of Nephrin was demonstrated by immunostaining, Western blotting and quantitative reverse transcriptase - polymerase chain reaction （qRT- PCR）. Results Most of the glomcruli were attached to culture dishes and exhibited cellular outgrowths, which were identified as podocytes by their distinct staining for Nephrin and WT - 1. The purity of podocytes was 94.7% according to flow cytometry analysis. The Nephrin expression levels of primary cultured podocytes were higher than those of subcultured cells. Conclusion The isolation method with collagenase provides a number of glomeruli suitable for primary podocyte culture with stable expression of Nephrin.

关 键 词：足细胞 细胞培养技术 NEPHRIN 

分 类 号：R692[医药卫生—泌尿科学]