中文摘要：

目的 体外观察不同时间高糖刺激的人肾小球系膜细胞(HGMC)中细胞衰老和自噬的变化,及自噬增强剂西罗莫司和自噬抑制剂3-甲基腺嘌呤(3MA)对高糖作用的影响.方法 体外培养HGMC,分别给予高糖(30.0 mmol/L葡萄糖)刺激12、24、48、72 h,及以500 nmol/L西罗莫司和2 mmol/L 3MA干预高糖培养72 h.设正常对照组(5.5 mmol/L葡萄糖)和高渗对照组(5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇).应用光镜观察细胞形态改变,透射电镜观察自噬体形成并计数,β半乳糖苷酶活性染色检测衰老细胞,Western印迹检测自噬相关蛋白LC3、p62及衰老相关蛋白p53、p21的表达.结果 与正常对照组比较,高糖组随着刺激时间延长逐渐表现出细胞体积变大、细胞质区域扁平、多形核及双核细胞增多等一系列细胞衰老表型,高糖刺激72 h时β半乳糖苷酶染色阳性率增加(P＜0.05);高糖刺激48 h和72 h时自噬小体数量均下降,LC3表达下降、p62、p53、p21蛋白表达升高(均P＜0.05).与高糖组比较,西罗莫司干预组LC3表达升高、p62、p53及p21表达下降,β半乳糖苷酶染色阳性率减少(均P＜ 0.05);而3MA干预组以上效应均无明显改变(均P＞0.05).结论 高糖体外刺激通过抑制自噬活性和激活p53/p21信号通路,可诱导HGMC衰老.西罗莫司增强自噬活性,可以抑制p53/p21通路蛋白表达,减轻衰老.

英文摘要：

Objective To observe the changes of senescence and autophagy in human glomerulus mesangial cells (HGMCs) induced by high glucose at different times,and to investigate the effects of rapamycin and 3-methyladenine (3-MA) on these changes.Methods HGMCs were cultured in vitro,exposed to high glucose (30.0 mmol/L glucose) for 12,24,48 and 72 h,and stimulated by high glucose with 500 nmol/L rapamycin or 2 mmol/L 3-MA for 72 h.Normal control group (5.5 mmol/L glucose) and hypertonic group (5.5 mmol/L glucose + 24.5 mmol/L mannitol) were set up.Cytomorphology changes were examined by light microscope to test whether cells were in senescent stage.The quantity of autophagosome was observed by electron microscope.The cell senescence was evaluated by β-galactosidase (SA β-gal) staining.The protein expressions of p53,p21,LC3 and p62 were determined by Western blotting.Results High glucose group gradually had larger size and more flat cytoplasm,polymorphonuclear cells and binucleate cells than control group as the stimulation times was prolonged.Compared with those in control group,SA β-gal positive cells in high glucose group after incubation for 72 h statistically were increased (P ＜ 0.05); the protein expressions of p62,p53 and p21 in high glucose group after incubation for 48 h and 72 h were increased (all P ＜ 0.05),while the autophagosome and the expression of LC3 decreased (P ＜ 0.05).Compared with those in high glucose group,the expression of LC3 was increased dramatically in high glucose with rapamycin group (P ＜ 0.05),while the protein expressions of p62,p53 and p21 decreased (all P ＜ 0.05),and SA β-gal positive cells decreased (P ＜ 0.05).However,there was no statistical difference between high glucose group and high glucose with 3-MA group in terms of above effects.Conclusions High glucose may induce HGMCs senescence through activating p53/p21 pathways and suppressing the activity of autophagy.Through enhanced autophagy activity with rapamycin,the expression of p53/p21 pathway was suppressed and senesc