氧化应激诱导卵巢颗粒细胞凋亡过程中调控线粒体自噬的关键因子研究  

Study on Key Factors Regulating Mitochondrial Autophagy During Oxidative Stress-induced Apoptosis in Ovarian Granulosa Cells

作　　者：杨礼莲 隋世燕 蒋蓉 徐琳杰 YANG Li-lian;SUI Shi-yan;JIANG Rong;XU Lin-jie(College of Public Health,Dali University,Dali,Yunnan 671000,China)

机构地区：[1]大理大学公共卫生学院,云南大理671000

出　　处：《动物医学进展》2025年第4期6-14,共9页Progress In Veterinary Medicine

基　　金：云南省科技人才与平台(专家工作站)计划(202305AF150083);国家自然科学基金项目(32260865);云南省高层次科技人才及创新团队选拔专项(202305AC160037);云南省科技厅基础研究计划(202101BA070001-202)。

摘　　要：为了筛选氧化应激诱导人卵巢颗粒细胞(KGN细胞)凋亡过程中参与线粒体自噬的关键蛋白,取对数生长的KGN细胞,分为对照组、过氧化氢(H_(2)O_(2))组、羰基氰化氯苯腙(CCCP)组,待细胞融合率达85%~90%时,H_(2)O_(2)组加入250μmol/L H_(2)O_(2)作用6 h建立氧化应激模型,CCCP组加入20μmol/L CCCP作用12 h建立线粒体自噬模型,对照组正常培养,收集细胞样本进行蛋白质组学分析,并使用蛋白免疫印迹(Western blot)法对关键蛋白进行验证。经生物信息学分析,结合参考文献,3个组之间有40个蛋白的表达存在差异,其中与线粒体自噬有关的蛋白有BNIP3、IRS2、GATA4和ENDOG;BNIP3与ENDOG存在相互联系。Western blot检测结果显示,BNIP3、IRS2和GATA4蛋白的表达水平与测序结果一致;与对照组相比,CCCP组ENDOG蛋白表达显著下调,但H_(2)O_(2)组无显著性差异。结果表明,BNIP3、IRS2和GATA4可能是氧化应激诱导KGN细胞凋亡过程中参与线粒体自噬的关键因子。In order to screen for key proteins involved in mitochondrial autophagy during oxidative stress-induced apoptosis in human ovarian granulosa cells(KGN cells),logarithmically grown KGN cells were taken and divided into the control group,hydrogen peroxide(H 2O 2)group,carbonyl cyanide m-chlorphenylhydrazone(CCCP)groups.When the cell fusion rate reached 85%-90%,250μmol/L H_(2)O_(2)was added to the H_(2)O_(2)group for 6 h to establish an oxidative stress model,and 20μmol/L CCCP was added to the CCCP group for 12 h to establish a mitochondrial autophagy model,and the control group was cultured normally,and cell samples were collected for proteomic analysis and protein immunoassay.Cell samples were collected for proteomic analysis,and the key proteins were verified by protein immunoblotting(Western blot).Bioinformatics analysis and references showed that the expression of 40 proteins differed among the three groups,among which the proteins related to mitochondrial autophagy were BNIP3,IRS2,GATA4,and ENDOG;among which,BNIP3 and ENDOG were interlinked.The results of the Western blot assay showed that the expression levels of BNIP3,IRS2,and GATA4 proteins were in agreement with the sequencing results.ENDOG protein expression was significantly down-regulated in the CCCP group compared with the control group,but there was no significant difference in the H_(2)O_(2)group.The results suggest that BNIP3,IRS2 and GATA4 may be the key factors involved in mitochondrial autophagy during oxidative stress-induced apoptosis in KGN cells.

关 键 词：人卵巢颗粒细胞 线粒体自噬 细胞凋亡 氧化应激 卵泡闭锁 

分 类 号：S852.1[农业科学—基础兽医学] S858.28[农业科学—兽医学]