缺氧微环境下3-甲基腺嘌呤对视网膜神经节细胞凋亡的影响  

Influence of 3-methyladenine in apoptosis of retinal ganglion cells under hypoxic micro-environment

作　　者：许文晶 王英[1] 杨海燕[1] 高志卓 徐韶琳[1] 

机构地区：[1]吉林大学第二医院青光眼科,吉林长春130041 [2]吉化集团公司总医院眼科,吉林吉林132013

出　　处：《吉林大学学报（医学版）》2014年第2期253-256,I0001,共5页Journal of Jilin University：Medicine Edition

基　　金：吉林省发展和改革委员会科研基金资助课题(JF2012C007-5)

摘　　要：目的:观察自噬抑制剂3-甲基腺嘌呤(3-MA)对视网膜神经节细胞(RGCs)凋亡的影响,阐明缺氧微环境下3-MA对RGCs的保护作用。方法:取对数生长期RGCs,分为对照组(PBS)、3-MA组(6mmol·L-13-MA)、CoCl2组(300μmol·L-1 CoCl2)、3-MA联合CoCl2组(6mmol·L-13-MA+300μmol·L-1 CoCl2)。MTT法检测CoCl2最适浓度和各组RGCs增殖抑制率;啶橙/溴化乙锭(AO/EB)染色法检测细胞形态;单丹(磺)酰戊二胺(MDC)染色法检测RGCs的自噬空泡;Western blotting法检测各组RGCs中凋亡蛋白P53、Bax、caspase-9、caspase-3、iASPP和自噬蛋白LC3表达水平。结果:CoCl2的30%抑制浓度(IC30)为300μmol·L-1。3-MA联合CoCl2组的细胞增殖抑制率低于CoCl2组(P<0.05)。荧光显微镜下观察,各组细胞出现数量不等的凋亡小体和自噬泡。Western blotting法检测,与对照组比较,其他3组RGCs中P53、Bax、caspase-9和caspase-3和LC3表达水平均增高(P<0.05),iASPP表达水平降低(P<0.05);3-MA联合CoCl2组的RGCs中P53、Bax、caspase-9和caspase-3和LC3表达水平低于CoCl2组(P<0.05),iASPP表达水平高于CoCl2组(P<0.05);3-MA联合CoCl2组RGCs中P53、Bax、caspase-9和caspase-3和LC3表达水平高于3-MA组(P<0.05),iASPP表达水平低于3-MA组(P<0.05)。结论:缺氧微环境下,自噬抑制剂3-MA对CoCl2诱导的RGCs凋亡具有保护作用。Objective To study the influence of the specific inhibitor of autophagy 3-methyladenine （3-MA）in apoptosis of retinal ganglion cells （RGCs）, and to clarify the protective effect of 3-MA on RGCs under hypoxie micro-environment. Methods The RGCs at logarithmic growth phase were collected and divided into control group （PBS）, 3-MA group （6 mmol· L^-1 3-MA）, COCl2 group（300 μmol·L^-1 COC12） and 3-MA combined with COC12 group（6 mmol · L^-1 3-MA＋ 300 μmol·L^-1 COC12）. MTT method was conducted to evaluate the optimal concentration of COC12 and the inhibitory rate of proliferation of RGCs in various groups; Acridine orange/ Ethidium bromide （AO/EB） staining was used to detect the morphology of RGCs; Monodansylcadaverine （MDC） staining was used to detect the autophagic vacuoles of RGCs; Western blotting method was used to test the expression levels of apoptotic proteins P53, Bax, caspase-9, caspase-3, iASPP and autophagic protein LC3 in RGCs in various groups. Results The 30% inhibitory concentration（IC30） of COC12 was 300 μmol·L^-1. Compared with COC12 group, the inhibitory rate of proliferation of RGCs in 3-MA＋CoC12 group was decreased（P〈0.05）. The apoptosis body and autophagic vacuole in RGCs in various groups were observed under fluorescence microscope. The Western blotting results showed that compared with control group, the expression levels of P53, Bax, caspase-9, caspase-3, and LC3 in RGCs in the other groups were increased（P^0.05） and the expression level of iASPP was decreased （P〈0.05）; compared with COC12 group, the expression levels of P53, Bax, caspase-9, caspase-3, and LC3 in RGCs in 3-MA -/-COCl2 group were decreased（P^0.05） and the expression level of iASPP was increased （P〈0.05）; compared with 3-MA group, the expression levels of P53, Bax, caspas-9, caspase-3, and LC3 in RGCs in 3-MA q-CoCl2 group were increased（P〈0.05） and the expression level of iASPP was decreased（P〈 0.05）. Conclusion Under hypoxic micro-environment,

关 键 词：视网膜神经节细胞 3-甲基腺嘌呤 自噬 细胞凋亡 缺氧 

分 类 号：R774.6[医药卫生—眼科]