大鼠足细胞原代培养及观察

Primary culture and observation of podocyte in vitro

目的建立一种操作简便、重复性较好的大鼠肾脏足细胞分离和培养方法 ,并观察体外培养足细胞的生长特性。方法选用SD大鼠幼鼠,无菌取肾,采用低浓度胰蛋白酶短时间消化结合差异过筛法,获得较高纯度的肾小球,接种于25cm2培养瓶,放置于37℃、5%CO2的恒温恒湿培养箱培养。采用细胞免疫组化技术,对所提取的细胞进行免疫染色鉴定,并在光学显微镜下对足细胞进行跟踪观察。结果培养第3天,可见有部分细胞从贴壁的肾小球爬出,第5天可见细胞成铺路石样生长,待第8天细胞汇合度达70%～80%,消化细胞传代培养,采用传代培养7 d的细胞进行免疫组化检测,细胞内足细胞标志蛋白Nephrin呈阳性表达,足细胞纯度高迏95%以上。足细胞生长良好,继续培养分化形成具有特殊形态的成熟足细胞。结论本实验成功建立了大鼠足细胞的提取、培养和鉴定方法,操作简单、重复性好、纯度高,为下一步足细胞相关研究奠定了基础。

[Objective]To establish an easy and repeatable method of isolation podocyte and observe characteristics of rats podocyte primary cultured in vitro. [Methods]The kidney was extracted from new natal Sprague-Dawley（SD）rat with sterile conditions. High pure glomerulars were seperated with combination low concentration of trypsin digestion in short time and different size of cell sieves. Then glomerular suspension was inoculated into 25 cm2 plastic flask coated with rat tail collagen and cultured with 37 ℃,5% CO2. Podocytes were identified by the cell immunohistochemistry staining（SP methods）and observed by optical microscope. [Results]The podocyte left from isolated glomeruli on the third day and grew with cobblestone-appearance at 5th day after primary culture. The podocytes were subcultured when it was confluent of 70%-80% at 8 th day. The intracellular Nephrin protein were positive expressed by immunohistochemistry staining. The purity of podocyte was over 95%. Podocytes grew well, and differentiated into mature cells which had special forms. [Conclusion]In this study, the extraction,culture and identification methods of rat podocytes were established successfully. The experimental methods with simple operation, repeatability and high purity laid the foundation for next podocyte-related research.