吉林大学学报(医学版)

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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

许文晶1, 王英1,杨海燕1,高志卓2,徐韶琳1   

  1. (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)
  • 收稿日期:2013-10-09 出版日期:2014-03-28 发布日期:2014-03-28
  • 通讯作者: 徐韶琳 E-mail:xushaolin2005@aliyun.com
  • 作者简介:许文晶（1986-），女，吉林省长春市人，在读医学硕士，主要从事青光眼视神经损害机制研究。
  • 基金资助:

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

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

XU Wen-jing1,WANG Ying1,YANG Hai-yan1,GAO Zhi-zhuo2,XU Shao-lin1    

  1. (1.Department of Glaucoma,Second Hospital ， Jilin University，Changchun 130041，China; 2.Department of Ophthalmology,Hospital of Jilin Chemical Industry Group,Jilin 132013，China)
  • Received:2013-10-09 Online:2014-03-28 Published:2014-03-28

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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(6 mmol?L-1 3-MA)、CoCl2组(300 μmol•L-1 CoCl2)、3-MA 联合CoCl2组(6 mmol?L-13-MA+300 μmol•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300　μmol•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。

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

Abstract:

Abstract: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  hypoxic 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 CoCl2) and 3-MA combined with CoCl2 group(6 mmol?L-13-MA+300 μmol•L-1 CoCl2).MTT method was conducted to evaluate the optimal concentration of CoCl2 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 CoCl2 was 300 μmol?L-1.Compared with CoCl2 group,the  inhibitory rate of proliferation of RGCs in 3-MA+CoCl2 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 CoCl2 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,caspase-9,caspase-3,and LC3 in RGCs in 3-MA +CoCl2 group were increased(P<0.05) and the expression level of  iASPP was decreased(P<0.05).Conclusion  Under  hypoxic micro-environment,the inhibitor of autophagy 3-MA has protective effect on apoptosis of RGCs induced by CoCl2.

Key words: retinal ganglion cells, 3-methyladenine, autophagy, apoptosis, hypoxia

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  • R774.6　
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