活性氧诱导血管平滑肌细胞衰老及脱氢表雄酮的干预研究

Vascular smooth muscle cell senescence induced by reactive oxygen species and intervention effect of dehydroepiandrosterone

目的观察三丁基过氧化氢（tert-butyl hydroperoxide，t—BHP）对血管平滑肌细胞（vascular smooth muscle cell，VSMC）衰老的诱导作用，及脱氢表雄酮（dehydroepiandrosterone，DHEA）的干预作用。方法将VSMC分为4组：对照组、t-BHP刺激组（在80μmol／L t-BHP的DMEM培养液中72h）；10nmol／LDHEA干预组（给予t-BHP刺激前30min先加用10nmol／LDHEA）；100nmol／LDHEA干预组（给予t-BHP刺激前30min先加用100nmol／LDHEA）。以衰老相关β-半乳糖苷酶活性和细胞的增殖能力两种衰老标志物为主要观察指标。其中衰老相关β-半乳糖苷酶活性采用免疫化学染色方法，流式细胞术检测细胞周期来反应细胞的增殖能力。结果经80mmol／L的t-BHP持续作用72h后，VSMC的G0／G1期细胞比例和SA-β半乳糖苷酶染色阳性细胞百分比增加[分别为（49．5±5．5）％和（89．4±3．4）％；（3．5±1．2）％和（75．3±4．3）％]，差异有统计学意义（P〈0．01），表明t-BHP成功诱导了VSMC衰老，而给予100nmol／LDHEA干预后上述变化改善，为（71．3±3．9）％。结论随增龄，活性氧损害的累积可能是VSMC衰老的机制之一，DHEA可能通过抗氧化作用延缓VSMC的衰老。

Objective To observe the onset of vascular smooth muscle cell （VSMC） senescence induced by tert-butyl hydroperoxide （t-BHP） and the intervention effect of dehydroepiandrosterone （DHEA）. Methods The VSMCs were divided into four groups： blank control group, t-BHP group （incubated with 80 btmol/L t-BHP for 72 h）, 10 nmol/L DHEA intervention group （pretreated with 10 nmol/L DHEA 30 min before t-BHP） and 100 nmol/L DHEA intervention group （pretreated with 100nmol/L DHEA 30 min before t-BHP）. Two ageing markers of ageing associated β-galactosidase activity and cell proliferation activity were adopted as main indexes. β-galactosidase activity was measured with immunocytoehemical method and cell proliferation activity was measured with flowcytometry. Results After continuous treatment with 80 mmol/L t-BHP for 72 h, the ratios of G0/G1 phase cells and SA-β-galactosidase staining positive cells increased as compared with blank controlgroup [（89.4±3.4）% vs. （49.5±5.5）%, （3.5±1.2）% vs. （75.3±4.3）%], which indicated that VSMCs senescence were successfully induced by t-BHP. While the above changes were smaller in 100 nmol/L DHEA intervention group than in t-BHP group. Conclusions With ageing, accumulation of damage produced by reactive oxygen species may be an important mechanism causing the onset of VSMCs senescence. DHEA may be able to retard the progression of VSMCs senescence through antioxidant effect.