鲍曼囊壁层上皮细胞培养与鉴定

Culture and identification of parietal epithelial cells of Bowman′s capsule

目的分离和培养小鼠鲍曼囊壁层上皮细胞,为进一步深入研究其生理作用提供基础。方法通过细胞筛联合磁性分离的方法分离小鼠肾小体,原代培养后获取壁层上皮细胞,诱导壁层上皮细胞分化为足细胞。使用免疫荧光染色、实时定量PCR和Western印迹法检测壁层上皮细胞和足细胞特异性标志物。结果原代培养的壁层上皮细胞呈现铺路石样,表达壁层上皮细胞特异性标志物Claudin-1和干细胞标志物CD133和CD24,不表达肾小管上皮细胞蛋白、系膜细胞和足细胞标志蛋白。体外培养传代至6代的壁层上皮细胞仍表达Claudin-1、CD133和CD24。经过诱导分化后,壁层上皮细胞可表达足细胞特异蛋白WT-1和Synaptopodin。结论细胞筛联合磁性分离法提取肾小体,体外培养获得的小鼠壁层上皮细胞可以在体外诱导表达足细胞标志蛋白。

Objective To explore the isolation and culture methods of mouse parietal epithelial cells(PECs)of Bowman′s capsule,so as to provide a cell tool for further study.Methods Mouse renal corpuscles were isolated by cell sieving combined with magnetic separation.After primary culture,identified parietal epithelial cells were induced to differentiate into podocytes.Immunofluorescence staining,real⁃time quantitative PCR and Western blotting were used to detect specific markers of parietal epithelial cells and podocytes.Results Primary cultured PECs grew like paving stone and expressed Claudin⁃1(PECs specific marker),CD133(stem cell marker)and CD24(stem cell marker),without the expression of tubular epithelial cell proteins,mesangial cell and podocyte specific proteins.Cultured to 6 generations in vitro,the PECs still expressed Claudin⁃1,CD133 and CD24.After incubated with differentiation medium,PECs were able to express podocyte markers WT⁃1 and Synaptopodin.Conclusion The renal corpuscles are extracted by cell sieving combined with magnetic separation,and the mouse PECs successfully cultured in vitro can be induced to express podocytes′markers.