庆大霉素诱导肾小管上皮细胞凋亡及细胞周期调节蛋白表达

Expression of cyclin E/CDK2 and p27Kipi in gentamicin-induced apoptosis of LLC-PKl cells

目的:观察庆大霉素对体外培养的肾小管上皮细胞凋亡的诱导作用;研究在细胞凋亡过程中细胞周期调节蛋白cy-clin E、CDK_2和p27^(Kipl)蛋白表达的改变,并探讨细胞凋亡与细胞周期调节蛋白表达之间的关系。方法:0.2～3.2 mg/ml的庆大霉素作用于体外培养的猪肾近端小管上皮细胞株(LLC-PK1细胞)24～96 h,MTT法测定庆大霉素对细胞增殖的抑制率,琼脂糖凝胶电泳法观察凋亡细胞DNA条带,流式细胞术测定细胞凋亡率(AI)和细胞增殖动力学,Western印迹分析检测培养96 h时细胞内cyclin E、CDK_2和p27^(Kipl)的蛋白表达。结果:庆大霉素对LLC-PK1细胞增殖有抑制作用,庆大霉素具诱导细胞凋亡的作用,AI呈药物浓度和作用时间依赖性;庆大霉素致细胞周期停滞于G_1期。随着庆大霉素浓度的增加,细胞内cy-clin E和 CDK_2蛋白表达逐渐下调,而p27^(Kipl)蛋白表达则逐渐上调。结论:庆大霉素诱导的LLC-PK1细胞凋亡与细胞周期调节蛋白cyclin E和 CDK_2表达下调以及p27^(Kipl)表达上调有关。

Objective:To observe the apoptosis of renal tubular epithelial cell line LLC-PK1 and expression levels of cell cycle regulatory protein cyclin E/CDK2 and p27Kip1 after being treated with gentamicin, and to probe into the relationship between apoptosis and cell cycle regulatory proteins. Methods:LLC-PK1 cells were treated with gentamicin (0. 2-3. 2 mg/ml) for 24-96 h. The growth inhibiting effect of gentamicin on cells were measured by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method. Agarose gel electrophoresis was performed to observe apoptosis of cells. Flow cytometry was used to determine the apoptotic indices (AT) of cells and distribution of cell cycle. Western Blotting was used to determine the expression of G1 phase-related regulatory protein cyclin E/CDK2 and p27Kip1 during the course of apoptosis. Results: Gentamicin inhibited the growth of LLC-PK1 cells. Agarose gel electrophoresis revealed the appearance of DNA ladder,a biochemical hallmark of apoptosis. AI increased linearly with the increase of gentamicin concentrations and times of incubation. Cell cycle analysis revealed G1 phase cells increased linearly with the increase of gentamicin concentrations. Western blotting showed that the protein levels of cyclin E and CDK2 were down-regulated, whereas those of p27Kip1 were up-regulated after treated with gentamicin. Conclusion: Gentamicin-induced apoptosis of LLC-PK1 cells is closely related to the increased expression of p27Kip1 and decreased expression of cyclin E and CDK2.