大鼠肾小球足细胞的原代培养与鉴定

Primary Culture and Identification of Rat Glomerular Podocytes

目的建立一种简单适用的原代培养与鉴定大鼠肾小球足细胞的方法。方法通过差异过筛法收获SD大鼠（体质量60～100g）肾小球，2g／L IV型胶原酶消化肾小球，组织块种植法将肾小球种植于添加有ITS-X（含转铁蛋白-亚硒酸钠-胰岛素）的K1—3T3培养基，9～10d后首次传代培养。通过观察细胞形态结合免疫组织化学SP法检测角蛋白、结蛋白和Wilms瘤抑癌因子-1（WT-1）表达以鉴定足细胞。结果种植肾小球3d后，可见从肾小球内生长出来的细胞逐渐增多，9～10d后融合成鹅卵石样外观。传代后的细胞变为大而扁平的星形细胞，有明显突起和微绒毛；免疫组织化学法显示结蛋白表达呈阴性、角蛋白和WT-1表达呈阳性，提示所培养的细胞为足细胞。结论种植胶原酶消化的肾小球是一种简单易行的原代培养大鼠肾小球足细胞方法。W-1可作为鉴定大鼠肾小球足细胞的合适标记。

Objective To establish an easy and feasible method for primary culture and identification of rat glomerular podocytes. Methods Glomeruli from Sprague-Dawley（SD） rats weighing 60-100 gram were isolated by the method of different size combination of screen. Isolated glomeruli were appropriately digested with 2 g/L type 1V collagenase and cultured in 25 cm2 plastic flask coated with rat tail collagen in K1-3T3 medium with ITS-X （containing insulin-transferrin-selenium）. Subculture of primary cultured epithelial cells was performed at 9-10 days after implantation of collagenase digested glomeruli. Podocytes were identified by the morphology study with scanning electron microscope and inverted microscope, as well as the immunohistocbemistry staining （SP methods） study for the expression of keratin, desmin and Wilms＇ tumor suppressor-1 （WT-1）. Results Epithelial cells outgrowth from isolated glomeruli appeared after 3 days primary culture and grew to confluence with cobblestone- appearance at 9-10 days. These cobblestone cells were subcultured at this point and gradually conversed into large, flat arborized cells with well-developed processes and microvilli. These arborized cells were negative expression with desmin staining and showed positive expression of cytokeratin and WT-1, which indicated that they were podocytes. Conclusion Implantating collagenase digested-glomeruli is an easy and feasible method for primary culture of rat glomerular podocytes. WT-1 may serve as a good marker to identify rat glomerular podocytes.