氧化应激诱导自噬对骨髓间充质干细胞增殖与凋亡的影响  

Effect of oxidative stress-induced autophagy on proliferation and apoptosis of MSCs

作　　者：刘关羽[1,2] 何卫阳[1] 朱鑫[1] 杨帆[1] 黄小龙[1,2] 印胡滨 苟欣[1] 

机构地区：[1]重庆医科大学附属第一医院泌尿外科 [2]分子肿瘤及表观遗传学重庆市重点实验室,重庆400016

出　　处：《中国病理生理杂志》2015年第12期2176-2182,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81370706;No.81372758);重庆市自然科学基金资助项目(No.cstc2013jcyj A10058);重庆市人力资源与社会保障局课题资助项目(渝留助2013011号);重庆市卫生局课题资助项目(No.2012-2-001)

摘　　要：目的:观察氧化应激是否可诱导骨髓间充质干细胞(MSCs)自噬,探索在氧化应激微环境中自噬对MSCs增殖和凋亡的影响及可能机制,以期提高移植MSCs治疗糖尿病勃起功能障碍(DMED)的效果。方法:以过氧化氢(H2O2)模拟氧化应激微环境。台盼兰染色细胞计数法及MTT分析检测不同浓度(0、50、100、200、400μmol/L)H2O2对MSCs增殖的影响。联合MTT、流式细胞术、Western blot及透射电镜等方法研究H2O2对MSCs凋亡及自噬的影响。结果:H2O2呈浓度依耐性抑制MSCs增殖,IC50=(384.5751±16.8895)μmol/L;Western blot分析及透射电镜提示H2O2在诱导MSCs凋亡的同时,诱导MSCs产生自噬;MTT分析及流式细胞术提示H2O2组细胞增殖受抑、凋亡率上升,阻断自噬后细胞增殖进一步减弱,凋亡率进一步上升;Western blot结果提示H2O2诱导cleaved caspase-3增多及多聚ADP-核糖聚合酶1(PARP1)裂解增强;阻断凋亡后,cleaved caspase-3降低及PARP1裂解减弱;阻断自噬后,cleaved caspase-3进一步增多及PARP1裂解进一步增强。结论:氧化应激对MSCs存活具有双重作用,在诱导MSCs凋亡的同时还诱导保护性自噬,阻断MSCs自噬可以增加MSCs的凋亡,因此进一步增强细胞保护性自噬将有望提高移植MSCs在糖尿病高氧化应激微环境中的存活率,从而改善移植MSCs对糖尿病勃起功能障碍的疗效。AIM： To investigate whether oxidative stress is able to induce autophagy in mesenchymal stem cells（ MSCs）,and to explore the effects of autophagy on MSC proliferation and apoptosis under oxidative stress circumstance as well as the underlying mechanism for promoting the therapeutic effects of transplanted MSCs on treating diabetes mellitus erectile dysfunction（ DMED）. METHODS： Hydrogen peroxide（ H2O2） was applied to simulate the oxidative stress circumstance. The effects of H2O2 at concentration of 0,50,100,200,400 μmol / L on the viability of MSCs were tested by the method of Trypan blue exclusion and MTT assay respectively. The methods of MTT assay,Western blot and transmission electron microscope（ TEM） were used to explore the effects of H2O2 on MSC apoptosis and autophagy. RESULTS：The proliferation of MSCs was obviously inhibited by H2O2 in a dose-dependent manner（ P〈0. 01） and the 50% inhibiting concentration（ IC50） was（ 384. 58 ± 16. 89） μmol/L. H2O2 induced apoptosis and autophay of MSCs. The proliferation rate of MSCs was suppressed by H2O2significantly（ P〈0. 05）,with a further decline by blockade of autophagy（ P〈0. 05） whereas increased by blockade of apoptosis（ P〈0. 05）. H2O2 induced MSCs apoptosis obviously（ P〈0. 05）,with an augment of apoptosis（ P〈0. 05） by blockade of autophagy. Furthermore,the H2O2 increased expression of cleaved caspase-3 and cleavage of poly ADP-ribose polymerase 1（ PARP1）,Which were decreased by apoptosis blockade whereas were enhanced by blockade of autopahgy. CONCLUSION： Oxidative stress plays a dual role in MSC survival,which induces MSC apoptosis and autophagy. Moreover,blockade of autophagy intensifies MSC apoptosis. Therefore,it is a promising method to ameliorate the effects of stem-cell based therapy on DMED by enhancing protective autophagy to increase the survival rate of transplanted MSCs against oxidative stress circumstance caused by diabetes mellitus.

关 键 词：自噬 间充质干细胞 细胞增殖 细胞凋亡 氧化应激 

分 类 号：R363.2[医药卫生—病理学] R698.1[医药卫生—基础医学]