人宫颈癌HeLa细胞氧化损伤中溶酶体与线粒体损伤的时间顺序及其意义  

Time order of lysosomal and mitochondrial cell death in oxidative injury of cervical cancer HeLa cells and its significance

作　　者：于春艳[1] 刘希[2] 于春荣 张钰[4] 苏静[4] 刘玉和[1] 

机构地区：[1]北华大学基础医学院病理学教研室,吉林吉林132013 [2]北华大学化学与生物学院实验中心,吉林吉林132013 [3]北京昭衍新药研究中心有限公司毒理部,北京100176 [4]吉林大学白求恩医学院病理生理学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2013年第5期872-875,I0002,共5页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金资助课题(81202552;81272876);吉林省教育厅科研基金资助课题(吉教科合字2011第117号;吉教科合字2012第394号);吉林省科技厅自然科学基金资助课题(201215103)

摘　　要：目的:探讨溶酶体及线粒体死亡途径在氧化应激诱导HeLa细胞凋亡过程中的时间顺序及其意义,阐明溶酶体膜通透性在氧化应激诱导线粒体凋亡过程中的重要作用。方法:以对数生长期人宫颈癌HeLa细胞作为研究对象,利用维生素K3(VK3)复制氧化应激模型,实验分为对照组、VK3 1h、VK3 3h、VK3 6h和VK312h作用组。MTT法检测各组细胞生存率;2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)染色检测细胞内活性氧(ROS)水平,吖啶橙(AO)染色观察溶酶体膜通透性,罗丹明123染色检测线粒体膜电势(ΔΨm),Western blotting法检测凋亡相关蛋白caspase-3活化片段的表达。结果:与对照组比较,VK3 6和12h组HeLa细胞生存率降低(P<0.05)。Western blotting法检测,与对照组比较,VK3 6和12h组HeLa细胞内caspase-3活化片段表达水平增加(P<0.05)。DCFH-DA染色,VK3作用1、3及6h,HeLa细胞内绿色荧光增强。AO染色,VK3 3h和6h组可见HeLa细胞的溶酶体内红色颗粒状荧光减弱,细胞浆绿色荧光增强。罗丹明123染色,VK36h组可见HeLa细胞浆内红色荧光强度明显降低(提示ΔΨm下降)。结论:氧化损伤能够诱导溶酶体及线粒体死亡途径,并且溶酶体的变化先于线粒体,提示溶酶体通透性在氧化应激诱导线粒体凋亡过程中具有重要作用。Objective To investigate the time order of lysosomal and mitochondrial cell death in human HeLa cells after oxidative stress injury and its significance,and to clarify the role of lysosomal membrane permeabilization in the process of mitochondrial apoptosis induced by oxidative stress.Methods The HeLa cells line was used for the study.Oxidative stress model of cervical cancer HeLa cells was established by vitamin K3（VK3）.The cells were divided into control,V3 1h,VK3 3h,VK3 6hand VK 12hgroups.The vitalities of HeLa cells in various groups were measured by MTT assay.The levels of reactive oxygen species（ROS）were determined by DCFH-DA.The lysosomal membrane permeabilization was observed by AO staining,and the mitochondrail membrane permeabilization in HeLa cells was detected by Rhodamine 123staining.The expression of apoptosis-related protein cleaved caspase-3was determined by Western blotting method.Results Compared with control group,the survival rates of HeLa cells in VK3 6hand VK3 12hgroups were decreased（P0.05）.Compared with control group,the cleaved caspase-3expression levels in VK 36hand VK3 12hgroups were increased detected by Western blotting method（P0.05）.The DCFH-DA results showed that after VK3treatment for 1h,3h,and 6h,the levels of green fluoresence were increased in HeLa cells.The AO staining results showed that VK3treatment for 3and 6h caused a decline in the red staining of lysosomes and increase in green staining of cytoplasm.The Rhodamine 123 results revealed that VK3treatment for 6 hinduced a drop in Rhodamine 123 red staining of cytoplasm.Conclusion Oxidative stress damage can induce lysosomal and mitochondrial cell death in HeLa cells,and lysosomal cell death eventuates ahead of mitochondrial,which prompting the lysosomal membrane permeabilization plays an important role in mitochondrial apoptosis pathway induced by oxidative stresss..

关 键 词：氧化应激 溶酶体 线粒体 细胞凋亡 

分 类 号：R363.2[医药卫生—病理学]