小鼠近端肾小管上皮细胞原代培养及鉴定

Primary culture and identification of mouse renal proximal tubular epithelial cells

本文旨在建立更有效的小鼠肾小管上皮细胞体外培养及鉴定方法。将小鼠肾脏皮髓质分离,取肾皮质将其充分剪碎,用II型胶原酶消化结合筛网过滤的方法获得小鼠近端肾小管上皮细胞,细胞培养在DMEM中。用倒置显微镜观察细胞形态及生长情况,用流式细胞仪检测细胞增殖能力,用CCK-8检测方法测定活力,用免疫荧光方法鉴定肾小管上皮细胞的纯度。结果显示,免疫荧光鉴定显示95%以上的细胞表达上皮细胞标志蛋白CK18,90%以上的细胞表达近端肾小管上皮细胞标志蛋白Villin、AQP1和SGLT2。细胞可传至第五代,随着传代次数增加,细胞增殖能力逐渐降低。以上结果提示,本研究成功建立了培养高纯度小鼠肾小管上皮细胞的方法,用这一改良方法获得的肾小管上皮细胞可用于后续的离体实验研究。

The present study was aimed to establish a modified method for culturing mouse renal proximal tubular epithelial cells （TECs）. Renal cortex was isolated from mouse kidney and scissored into pieces. TECs were separated by digesting scissored renal cortex in type II collagenase combined with strainer filtration, and then cultured in DMEM. The morphology of TECs was observed under inverted microscopy. The cell proliferative ability was assessed by flow cytometry, and cell viability was analyzed by CCK-8 assay. The purity of TECs was identified by immunofluorescence. Immunofluorescence observation showed that more than 95% cells were epithelial marker CK18 positive and more than 90% cells expressed renal proximal TECs marker proteins, Villin, AQP1, and SGLT2. The cells could be subcultured for about 5 times. The cell proliferative ability declined following the repeated passage. This study introduced a modified efficient method for culturing highly purified mouse renal proximal TECs.