大鼠肾小球系膜细胞原代培养与鉴定

The Primary Culture of Glomerular Mesangial Cell from Rats and Its Identification

目的:原代培养及鉴定大鼠肾小球系膜细胞(GMC),探索分离、培养GMC的最佳条件。方法:对大鼠双肾灌洗后,筛网滤过分离肾小球,并用Ⅳ型胶原酶消化处理,然后种植法体外培养大鼠肾小球。同时采用免疫细胞化学和免疫荧光技术,检测亚代GMC的α-平滑肌肌动蛋白(α-SMA)、波形蛋白(V im entin)、细胞角蛋白(Cytokeratin)、Ⅷ因子相关抗原抗体的表达,鉴定GMC,并绘制其生长曲线。结果:种植4 d后,约80%肾小球贴壁,可见卵圆形上皮细胞生长,18 d左右镜下细胞多呈星状或三角形并伴有不规则的突起,21 d后细胞生长密集,细胞团簇之间形成网络。免疫细胞化学和免疫荧光证实抗α-SMA及抗V im entin染色阳性,而抗Cytokera-tin、抗Ⅷ因子抗体染色为阴性。GMC亚代倍增时间为4 d。结论:结果表明,用改良的培养方法成功率高,培养的细胞为大鼠肾小球系膜细胞。

Objective： To culture and identify the glomerular mesangial cells （GMC） of rats. The optimum conditions for culturing of GMC were also investigated. Methods： After all kidneys of eight normal SD rats were douched, glomeruli were separated using a three-layer micropore filter device and digested with type Ⅳ collagenase. The treated glomeruli were cultured in MEM. The cellular markers including α-SMA, vimentin, cytokeratin and factor Ⅷ were detected with immunocytochemistry and immunofluorescence methods, in order to identify GMC and draw the growth curve. Results： Round cells in culture began to grow on day 4, and then, the cells appeared stellate or triangular with irregular cytoplasmic projections in about 18 days observed through light microscope. The immunocytochemistry stainings of α-SMA and vimentin were positive in the cultured glomerular mesangial cells, while those of factor Ⅷ and cytokeratin negative. The doubling time of the sub-generation of GMC was 4 days. Conclusions： The results suggest that this improved cell culture method be successful and the cultured cells from the glomeruli of rats were glomerular mesangial cells.