缺血缺氧对VX2细胞自噬活性和生物学行为的影响  

Effects of ischemia and hypoxia on autophagy activity and biological behavior of VX2 cells

作　　者：张延超 刘旺根 王朝阳[1] 魏乐群 王彤 肖全平[1] 杜廷伟[1] 杨海 牛焕章 ZHANG Yanchao;LIU Wanggen;WANG Zhaoyang;WEI Lequn;WANG Tong;XIAO Quanping;DU Tingwei;YANG Hai;NIU Huanzhang(Department of Interventional Radiology,the First Affiliated Hospital,and College of Clinical Medicine of He'nan University of Science and Technology,Luoyang,He'nan Province 471003,China;Department of Histology and Embryology,Preclinical Medicine College,Xinxiang Medical University,Xinjiang nan Province 453003,China)

机构地区：[1]河南科技大学临床医学院河南科技大学第一附属医院介入科,河南洛阳471003 [2]新乡医学院基础医学院组织学与胚胎学教研室,河南新乡453003

出　　处：《实用放射学杂志》2021年第6期1017-1021,共5页Journal of Practical Radiology

摘　　要：目的研究自噬在不同阶段对VX2细胞增殖、侵袭和转移的影响.方法将VX2细胞分为对照组、3-甲基嘌呤(3-MA)组、缺血缺氧(I/H)组、I/H+3-MA组、再灌注组、再灌注+3-MA组.荧光显微镜观察各组细胞吖啶橙染色后细胞质内自噬泡数量;Western blot检测LC3及P62(P62/SQSTM1)信号蛋白、基质金属蛋白酶MMP2、MMP9蛋白表达情况;MTT实验测定细胞存活情况;细胞划痕实验观察迁移能力;Transwell实验评价VX2细胞迁移及侵袭能力.结果缺血缺氧早期吖啶橙标记的橙色或红色自噬泡增多,LC3Ⅱ/LC3Ⅰ表达增大、P62表达减少,加入3-MA后细胞存活数目由0.38±0.01减至0.25±0.01(P<0.05);I/H后、再灌注后LC3Ⅱ/LC3Ⅰ表达进一步增大(P<0.01),同时MMP2、MMP9蛋白表达也增加,侵袭性细胞计数高于对照组(121.0±5.2 vs 78.3±7.3,P<0.01);加入3-MA后细胞存活数目由0.50±0.01增加到0.61±0.01(P<0.05),侵袭性细胞计数高于再灌注+3-MA组(121.0±5.2 vs 70.0±2.1,P<0.05).结论缺血缺氧可诱导VX2细胞产生保护性自噬;再灌注后自噬过度激活,对细胞有杀伤作用,联合3-MA抑制自噬细胞存活增多、侵袭性减低;再灌注后VX2肝癌细胞自噬增强促进VX2细胞迁移与侵袭,这可能是局部浸润和远处转移的机制之一,可能为肝动脉化疗栓塞(TACE)术后肝细胞癌(HCC)的复发、转移治疗提供理论基础.Objective To study the effect of autophagy on proliferation,invasion and metastavsis of VX2 tumor cells at different stages.Methods The VX2 cells were randomly divided into six groups:control group,3-MA group,ischemia/hypoxia(I/H)group,I/H-3-MA group,repcrfusion group,and reperfusion+3-MA group.The fluorescence microscopy was used to observe the red or orange-red acidic vesicles in cytoplasm after acridine orange staining.Expressions of autophagy specific protein LC3 and P62(P62/SQSTM1)signaling protein,matrix metalloproteinase MMP2 and MMP9 were detected by Western blot.The cell survival was measured by MTT assay,and cell migration ability was observed by the scratch experiment.Transwell assay was performed to evaluate the migration and invasion ability of VX2 cells after ischemia and hypoxia.Results The acridine orange markers increased in the number of orange or red autophagy bubble in the early ischemic anoxia.LC3Ⅱ/LC3Ⅰexpression was also increased,and P62 expression was decreased.After addition of 3-MA,the number of cells alive decreased from 0.38±0.01 to 0.25±0.01(P<0.05).LC3Ⅱ/LC3Ⅰexpression further increased after I/H and reperfusion(P<0.01),at the same time in the MMP2 and MMP9 protein expressions increased.The number of invasive cells was higher than that of control group(121.0±5.2 vs 78.3±7.3,0.01).When 3-MA was added,the number of survived cells increased from 0.50±0.01 to 0.61±0.01(P<0.05).The invasive cell count was higher than that of the reperfusion+3-MA group(121.0±5.2 vs 70.0±2.1,P<0.05).Conclusion Ischemia and hypoxia can induce protective autophagyin VX2 hepatoma cells.After reperfusion,the autophagy is overactivated,with a killing effect on cells.When 3-MA is added,the survival of cells is increased and the invasiveness is decreased.Enhancement of VX2 promotes cells migration and invasion after reperfusion,which may be one of the mechanisms of local infiltration and distant metastasis.This may provide a theoretical basis for the treatment of hepatocellular carcinoma(HCC)recurre

关 键 词：自噬 肝细胞癌 缺氧 缺血 肝动脉化疗栓塞 

分 类 号：R735.7[医药卫生—肿瘤] R815[医药卫生—临床医学]