大鼠肾小管上皮细胞的分离鉴定及其增殖活力分析

目的：建立一种大鼠肾小管上皮细胞的分离鉴定方法，分析所获得细胞的增殖活力，为肾损伤等肾脏疾病的体外分析实验提供理论基础和技术支持。方法：采用Ⅱ型胶原酶消化法分离肾小管节段，并用Percoll密度梯度离心法进一步纯化肾小管，常规条件下对肾小管节段进行贴壁培养，待细胞爬出后，用免疫组织化学方法检测细胞角质素-18（CK-18）的表达情况，采用流式细胞术对第0、1和2（P0、P1、P2）代细胞的CK-18表达情况进行分析，用CCK-8方法检测各代细胞的增殖活力。结果：肾小管节段培养24 h后，可见细胞从节段内爬出；培养72 h后，细胞呈铺路石样密集生长。随着传代进行，P0、P1和P2代细胞CK-18的表达效率分别为95.9%、91.5%和76.8%，肾小管上皮细胞逐渐死亡，P2代细胞较P1代细胞增殖活力明显减弱(P<0.05)，同时培养体系中杂细胞增多。结论：该方法经济可靠、简便易行，但是所分离的肾小管上皮细胞只可在体外传代2次，不能长期培养，仅P0、P1代细胞可供体外分析实验使用。

Separation and identification of rat renal tubular epithelial cells and analysis of its proliferation ability

Objective To establish a method to separate and identify the rat renal tubular epithelial cells(RTECs) and analyze the proliferation ability of the obtained cells,and to provide theoretical basis and technique support for analysis experiment in vitro of kidney diseases such as kidney injury and so on.Methods The renal tubules were separated with type Ⅱ collagenase digestion and purified with Percoll density gradient centrifugation and cultured in normal environment,when the RTECs grew out from the tubule,the cytokeratin-18(CK-18) expression was detected with immunohistochemistry and the expressions of CK-18 in P0,P1 and P2 cells were detected with flow cytometry.The proliferation ability of each generation was evaluated with CCK-8 method.Results After cultured for 24 h,the RTECs began to grow outside from the tubule and they looked like paving road stones after cultured for 72 h.In the process of passage,the expression efficiencies of CK-18 were 95.9%,91.5%,and 76.8% in P0,P1 and P2 RTECs,and the RTECs were dead gradually.The proliferation ability of P2 cells was more weaken than that of P1 cells(P<0.05),and more mixed cells existed in the culture system.Conclusion The method established in this study is cheap,credited as well as convenient.However,the cells just could be passaged for 2 times and couldn't be cultured for long time.Only the cells of P0 and P1 could be applied in analysis experiment in vitro.