肾小球系膜细胞的分离和培养

Isolation,identification and in vitro culture of human glomerular mesangial cells

目的分离、培养和鉴定正常人肾小球系膜细胞。方法筛网滤过分离正常人肾小球系膜细胞,同时采用免疫荧光技术,利用异硫氰酸荧光素(FITC)间接标记单克隆抗体:抗Ⅷ因子相关抗原抗体、抗结蛋白抗体、抗角蛋白抗体、抗细胞角蛋白抗体、抗Ⅳ型胶原抗体、抗纤维连接蛋白抗体、抗层粘连蛋白抗体对系膜细胞特有的中间微丝进行鉴定。并采用同期培养的同一来源的肾小球内皮细胞作对照。结果抗Ⅷ因子相关抗原、抗角蛋白和抗细胞角蛋白抗体表达均为阴性,而抗结蛋白抗体、抗Ⅳ型胶原抗体、抗纤维连接蛋白抗体、抗层粘连蛋白抗体为阳性,说明培养的细胞为系膜细胞。结论该种方法分离培养的细胞经过免疫荧光染色鉴定为肾小球系膜细胞。

Objective To define the optimal condition to culture human glomerular mesangial cells in vitro. Methods With a three-layer micropore filter device, we separated the glomerular mesangial cells from normal human, with were subjected to identification with immunohistochemical method. Fluorescein isothiocyanate (FITC) staining was performed for labeling of the monoclonal antibodies so as to identify the cells' superficial markers including factor Ⅷ, keratin, cytokeratin, desmine, et al. Glomerular endothelial cells isolated from the same human tissues were also cultured in vitro under the same condition, serving as negative control. Results and conclusion The antibodies related to anti-factor Ⅷ, and those of antikeratin and anticytokeratin were negative in the isolated glomerular mesangial cells, while the antibodies for desmine, anti-collagen Ⅳ, anti-fibronectin, and anti-laminin were positive, offering evidence for confirming the identification of the isolated and cultured mesangial cells.