目的 研究萬(wàn)古霉素誘導(dǎo)人腎小管上皮細(xì)胞（HK-2）損傷及其Smad同源物4（Smad4）表達(dá)，并探討其潛在的作用機(jī)制。方法 使用不同終濃度（0、1、2、3、4、5 mmol/L）的萬(wàn)古霉素溶液作用于HK-2細(xì)胞24 h，顯微鏡下觀察萬(wàn)古霉素對(duì)細(xì)胞形態(tài)的影響；蛋白質(zhì)免疫印跡法檢測(cè)Smad4、B淋巴細(xì)胞瘤-2（Bcl-2）相關(guān)X蛋白（Bax）、Bcl-2的蛋白水平；TUNEL法檢測(cè)HK-2細(xì)胞凋亡情況；CCK8法檢測(cè)HK-2細(xì)胞活力來(lái)研究萬(wàn)古霉素對(duì)腎臟的毒性作用。構(gòu)建Smad4過(guò)表達(dá)質(zhì)粒，通過(guò)CCK8法和TUNEL實(shí)驗(yàn)檢測(cè)Smad4過(guò)表達(dá)對(duì)萬(wàn)古霉素誘導(dǎo)的HK-2細(xì)胞損傷的影響。結(jié)果 與對(duì)照組比較，隨著萬(wàn)古霉素濃度升高，HK-2細(xì)胞逐漸變形，數(shù)量逐漸減少；細(xì)胞活力顯著降低，凋亡率顯著增加（P＜0.05、0.01）。與對(duì)照組比較，促凋亡蛋白Bax表達(dá)顯著增高，而抗凋亡蛋白Smad4、Bcl-2表達(dá)則顯著降低（P＜0.05、0.01、0.001）。過(guò)表達(dá)Smad4后，使萬(wàn)古霉素誘導(dǎo)的HK-2的細(xì)胞活力顯著增加，凋亡率顯著降低（P＜0.05、0.001）。結(jié)論 萬(wàn)古霉素能夠誘導(dǎo)人腎小管上皮細(xì)胞損傷，其作用機(jī)制可能與調(diào)控Smad4的蛋白降解密切相關(guān)。

Objective To study the injury of human renal tubular epithelial cells (HK-2) induced by vancomycin and the expression of Smad4, and to explore the potential mechanism of action. Methods HK-2 cells were treated with vancomycin solution at different final concentrations (0, 1, 2, 3, 4, and 5 mmol/L) for 24 h, and the effect of vancomycin on cell morphology was observed under microscope. The protein levels of Smad4, Bax, and Bcl-2 were detected by Western blotting. The apoptosis of HK-2 cells was detected by TUNEL method. The activity of HK-2 cells was detected by CCK8 method to study the toxic effect of vancomycin on kidney. Smad4 overexpression plasmid was constructed, and the effect of Smad4 overexpression on vancomycin-induced HK-2 cell damage was detected by CCK8 assay and TUNEL assay. Results Compared with the control group, with the increase of vancomycin concentration, the number of HK-2 cells was gradually deformed and decreased. The cell viability was significantly decreased and the apoptosis rate was significantly increased (P＜ 0.05, 0.01). Compared with the control group, the expressions of pro-apoptotic protein Bax were significantly increased, while the expressions of anti-apoptotic proteins Smad4 and Bcl-2 were significantly decreased (P＜ 0.05, 0.01, 0.001). After overexpression of Smad4, vancomycin-induced HK-2 cell viability was significantly increased, and apoptosis rate was significantly decreased (P＜0.05, 0.001). Conclusion Vancomycin can induce the injury of human renal tubular epithelial cells, and its mechanism may be closely related to the regulation of Smad4 protein degradation.

R965

天津市濱海新區(qū)衛(wèi)健委科技項(xiàng)目（2019BWKQ029）