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目的:采用庆大霉素、顺铂、马兜铃酸Ⅰ诱导兔原代肾小管上皮细胞构建体外肾毒性细胞模型。方法:分离培养兔原代肾小管上皮细胞(RTECs),酶化学染色和免疫组化鉴定;采用MTT法绘制兔RTECs传代培养生长曲线并确定最佳接种细胞量及加药时间;测定不同浓度庆大霉素、顺铂、马兜铃酸Ⅰ对细胞增殖作用及乳酸脱氢酶(LDH)释放量的影响。结果:碱性磷酸酶染色及免疫组化染色均呈阳性结果证明获得兔RTECs,细胞传代培养的最佳接种量为1×104个/孔,最佳给药时间为接种48h。不同浓度庆大霉素、顺铂、马兜铃酸Ⅰ均使细胞形态有一定的改变,对细胞增殖均有抑制作用,且呈浓度依赖性。三者均使细胞LDH的释放量增高,呈浓度依赖性,其中顺铂和马兜铃酸Ⅰ的作用最显著。结论:采用庆大霉素、顺铂、马兜铃酸Ⅰ可诱导兔原代肾小管上皮细胞构建体外肾毒性细胞模型。
OBJECTIVE: To establish an in vitro model of nephrotoxicity induced by gentamicin, cisplatin and aristolochic acid Ⅰ in primary renal tubular epithelial cells in rabbits. Methods: Rabbit primary renal tubular epithelial cells (RTECs) were isolated and cultured, and the enzyme staining and immunohistochemical staining were used for identification. The growth curve of subcultured rabbit RTECs was drawn by MTT method and the optimal amount of inoculated cells and dosing time were determined. Effects of gentamicin, cisplatin and aristolochic acid Ⅰ on cell proliferation and lactate dehydrogenase (LDH) release. Results: The positive results of alkaline phosphatase staining and immunohistochemical staining showed that rabbit RTECs were obtained. The optimal inoculum size was 1 × 104 cells / well, and the best time of inoculation was 48h. Different concentrations of gentamicin, cisplatin, aristolochic acid Ⅰ have some changes in cell morphology, have an inhibitory effect on cell proliferation, and in a concentration-dependent manner. All three cells increased the release of LDH in a concentration-dependent manner, of which cisplatin and aristolochic acid Ⅰ had the most significant effect. Conclusion: Gentamycin, cisplatin and aristolochic acid Ⅰ can induce primary renal tubular epithelial cells in vitro to establish nephrotoxicity model.