小鼠肾脏足细胞的原代培养和鉴定

目的：建立可操作性好、简单易行且效率较高的小鼠肾脏足细胞分离及原代培养模式，为其进一步研究奠定基础。方法：取C57/BL6J小鼠肾脏，通过差异过筛法获取300目筛的肾小球，用制备好的KI-3T3培养基重悬肾小球后静置于铺被鼠尾胶原的培养皿中，4 d后开始换液，7 d后开始胰蛋白酶消化肾小球，并开始足细胞传代培养。5~7d传代1次，传代2~3次后收集细胞鉴定。采用倒置扭转显微镜观察小鼠肾脏足细胞形态表现，采用PCR法检测小鼠肾脏足细胞中nephrin，podocin和P-cadherin的表达。结果：肾小球种植3 d后可见足细胞从组织中爬出，足细胞传代培养7 d后在倒置相差显微镜下可观察到细胞胞体有突起生长。PCR法检测，分离培养的足细胞表达nephrin、podocin和P-cadherin。结论：差异过筛结合消化酶技术能够成功分离培养C57/BL6J小鼠肾脏足细胞。

Primary culture and identification of mouse kidney cells

Objective: To establish a good operability, simple and efficient mouse kidney cell separation and primary culture model, and to lay the foundation for further study.Methods: The murine kidney tissues were collected and the glomeruli with different size combination of screening were obtained. The isolated glomeruli were suspensioned in KI-3T3 medium, then subsided in the medium cell with rat tail collagen. The glomeruli were refreshed 4 d after cultivation. 7 d after cultivation, the glomeruli were resolved by trypsin and the podocyts were cultured. The podocytes were generated at 5-7 d. The podocytes were analyzed after 2-3 generations. The morphology of podocytes of kidney tissue of the mice was observed by inverted phase contract microscope.The expressions of nephrin,podocin and P-cadherin in the podocytes of kidney tissue of the mice were detected by PCR.Results: The podocytes began to remove from the planted glomeruli at the 3th day. After 7 d of generation, the cultured podocytes showed microvilli and process.The PCR results showed that the podocytes expressed nephrin,podocin, and P-cadherin.Conclusion: The difference sifting technology combined with enzyme digestion could successfully isolate and culture the C57/BL6J mouse podocytes in vitro.