【摘要】 目的:探讨雷帕霉素（Rapa）对过氧化氢（H₂O₂）诱导的人脐静脉内皮细胞（HUVECs）衰老的影响及其可能的机制。方法:把HUVECs分为空白组、衰老组（H₂O₂诱导）、Rapa+H₂O₂组和3-甲基腺嘌呤（3-MA）+H₂O₂组。噻唑蓝（MTT）比色法检测各组细胞活力;衰老相关β-半乳糖苷酶（SA-β-Gal）染色检测细胞衰老情况;透射电子显微镜观察细胞超微结构改变;Western blot检测Rb、磷酸化Rb（p-Rb）、p21、LC-3II和beclin-1的表达变化。结果:与空白组相比,衰老组细胞活力下降,SA-β-Gal染色阳性率增高,细胞结构紊乱,并伴有衰老蛋白表达显著增加（P<0.01）;与衰老组相比,Rapa+H₂O₂组细胞活力增强,细胞衰老染色减少,p-Rb和p21表达显著减少（P<0.05）,电镜下细胞结构趋于正常,并可见自噬小体,beclin-1和LC3-II表达显著增多（P<0.01）;与衰老组相比,给予自噬抑制剂3-MA的细胞则表现为细胞活力进一步下降,SA-β-Gal染色阳性率进一步增高,p-Rb和p21表达增多,细胞结构破坏明显,beclin-1和LC3-II几乎不表达（P<0.05）。结论:Rapa可延缓H₂O₂诱导的血管内皮细胞衰老,这种抗衰老作用可能与促进自噬有关。更多还原

【Abstract】 AIM: To investigate the effects of rapamycin（Rapa） on hydrogen peroxide（H₂O₂）-induced vascular endothelial cell senescence and to explore the underlying mechanisms. METHODS: The human umbilical vascular endothelial cells（HUVECs） were divided into 4 groups: control group, senescence group, Rapa+H₂O₂ group and 3-methyladenine（3-MA）+H₂O₂ group. MTT assay was performed to assess the cell viability. Senescence-associated β-ga-lactosidase（SA-β-Gal） staining was performed to measure the senescent cells in each group. The subcellular structures were observed under transmission electron microscope（TEM）. The protein levels of phosphorylated Rb（p-Rb）, Rb, p21, LC3-II and beclin-1 were determined by Western blot. RESULTS: Compared with control group, the cell viability in H₂O₂ group was significantly decreased accompanied with higher rate of SA-β-Gal staining positive cells（P<0.05） and markedly damaged structure. Additionally, the protein levels of p-Rb and p21 in senescence group were increased markedly compared with control group（P<0.05）. However, the cells pre-treated with Rapa prior to stimulation with H₂O₂ showed increased viability, decreased number of senescent cells and decreased protein levels of p-Rb and p21 as compared with the cells stimulated with H₂O₂ alone（P<0.05）. Moreover, the TEM observation showed that the structure of the cells in Rapa+H₂O₂ group was roughly normal and the autophagosome was captured, and the expression levels of beclin-1 and LC3-II were increased（P<0.05）. Conversely, pre-treatment with autophagy inhibitor 3-MA resulted in opposite results. The cell viability was decreased significantly, more senescent cells were stained blue, higher protein levels of p-Rb and p21 were detected（P<0.05）, poor subcellular structures were captured, and no beclin-1 and LC3-II was detected. CONCLUSION: Rapa may retard the senescence of HUVECs induced by H₂O₂, and promoting autophagy may be the underlying mechanism.更多还原