小鼠肾脏足细胞的原代培养和鉴定  

Primary culture and identification of mouse kidney cells

作　　者：高霞[1] 雷晓燕[1] 刘姝娆 索艳红 曹晓锋[1] 高明东[1] 

机构地区：[1]甘肃省人民医院儿科,甘肃兰州730000 [2]甘肃省中医药大学研究生处,甘肃兰州730000 [3]宁夏医科大学研究生学院,宁夏银川750000

出　　处：《吉林大学学报（医学版）》2017年第1期186-189,I0004,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金地区项目资助课题(81360114);甘肃省科技厅技术研究与开发专项计划资助课题(1305TCYA035);甘肃省卫计委行业项目资助课题(GSWSKY-2015-12);甘肃省委组织部陇原青年创新人才扶持计划资助课题[甘组通字(2014)4号]

摘　　要：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,4d后开始换液,7d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3d后可见足细胞从组织中爬出,足细胞传代培养7d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。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.

关 键 词：肾脏 足细胞 原代培养 

分 类 号：Q813.11[生物学—生物工程]