沉默FOXO1基因对人主动脉血管平滑肌细胞自噬和凋亡的影响  

Effect of silencing FOXO1 gene on autophagy and apoptosis of human aortic vascular smooth muscle cells

作　　者：王琳茹 张晶[1] 赵冬婵 王晋军[1] 胡文贤 WANG Linru;ZHANG Jing;ZHAO Dongchan;WANG Jinjun;HU Wenxian(Department of Vascular Surgery,Affiliated Hister Hospital of Qingdao City,Qingdao University,Traditional Chinese Medicine Hospital of Qingdao City,Qingdao 266000,China)

机构地区：[1]青岛大学附属青岛市海慈医院青岛市中医医院血管外科,山东青岛266000

出　　处：《吉林大学学报（医学版）》2024年第2期431-441,共11页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(82172095);山东省科技厅自然科学基金项目(ZR2022ME083);山东省卫健委中医药科技发展计划项目(2019-WJZD045);山东省卫健委医药卫生科技发展项目(202104130109)。

摘　　要：目的:探讨叉头框转录因子O1(FOXO1)基因对腹主动脉瘤(AAA)血管平滑肌细胞自噬和凋亡的影响,阐明其可能的作用机制。方法:收集19例AAA患者动脉瘤组织(AAA组)及邻近正常主动脉组织(对照组),采用实时荧光定量PCR(RT-qPCR)法检测2组研究对象动脉瘤组织中FOXO1 mRNA表达水平,透射电镜观察2组研究对象动脉瘤组织中自噬溶酶体形成情况;Western blotting法检测2组研究对象动脉瘤组织中FOXO1及自噬相关蛋白卷曲螺旋肌球蛋白样B细胞淋巴瘤2(Bcl-2)结合蛋白(Beclin1)、微管相关蛋白1轻链3α(LC3)和P62蛋白表达水平。体外培养人主动脉血管平滑肌细胞(hVSMCs),并采用FOXO1 siRNA(si-FOXO1)及其阴性对照(si-NC)慢病毒感染hVSMCs,10μmol·L^(-1)血管紧张素Ⅱ(AngⅡ)联合自噬激活剂雷帕霉素(Rap)进行干预,将细胞分为空白对照组、AngⅡ组、AngⅡ+si-NC组、AngⅡ+si-FOXO1组、AngⅡ+si-NC+Rap组和AngⅡ+si-FOXO1+Rap组。CCK-8法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡水平,ELISA法检测各组细胞上清中基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)水平,RT-qPCR法检测各组细胞中FOXO1 mRNA表达水平,Western blotting法检测各组细胞中FOXO1、Bcl-2、Bcl-2相关X蛋白(Bax)、剪切型含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)、Beclin1、LC3和P62蛋白表达水平。结果:与对照组比较,AAA组动脉瘤组织中FOXO1 mRNA表达水平升高(P<0.05),自噬溶酶体数量增多(P<0.05),Beclin1蛋白表达水平和LC3Ⅱ/LC3Ⅰ比值升高(P<0.05),P62蛋白表达水平降低(P<0.05)。与空白对照组比较,AngⅡ组hVSMCs增殖活性降低(P<0.05),细胞凋亡率升高(P<0.05),细胞上清中MMP-2和MMP-9水平升高(P<0.05),细胞中Bax、Cleaved caspase-3和Beclin1蛋白表达水平及LC3Ⅱ/LC3Ⅰ比值升高(P<0.05),Bcl-2和P62蛋白表达水平降低(P<0.05);与AngⅡ+si-NC组比较,AngⅡ+si-FOXO1组hVSMCs增殖活性升高(P<0.05),细胞凋亡率降低(Objective:To discuss the effect of forkhead box O1(FOXO1)gene on the autophagy and apoptosis of the vascular smooth muscle cells in abdominal aortic aneurysm(AAA),and to clarify its possible mechanism.Methods:The aneurysm tissue of nineteen AAA patients(AAA group)and adjacent normal aortic tissue of nineteen AAA patients(control group)were collected.Real-time fluorenscence quantitative PCR(RT-qPCR)method was used to detect the expression level of FOXO1 mRNA in aneurysm tissue of the subjects in two groups;transmission electron microscope was used to observe the autophagolysosome formation in aneurysm tissue of the subjects in two groups;Western blotting method was used to detect the expression levels of FOXO1 and autophagy-related proteins B cell lymphoma-2(Bcl-2)-binding protein(Beclin1),microtubule-associated protein 1 light chain 3α(LC3),and P62 proteins in aneurysm tissue of the subjects in two groups.The human aortic vascular smooth muscle cells(hVSMCs)were cultured in vitro and infected with FOXO1 siRNA(si-FOXO1)and its negative control(si-NC)lentivirus,then treated with 10μmol·L^(-1) angiotensinⅡ(AngⅡ)combined with autophagy activator rapamycin(Rap).The cells were divided into blank control group,AngⅡgroup,AngⅡ+si-NC group,AngⅡ+si-FOXO1 group,AngⅡ+si-NC+Rap group,and AngⅡ+si-FOXO1+Rap group.CCK-8 assay was used to detect the proliferation activities of the cells in various groups;flow cytometry was used to detect the apoptotic rates of the cells in various groups;ELISA method was used to detect the levels of matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9)in the cell supernatant in various groups;RT-qPCR method was used to detect the expression level of FOXO1 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of FOXO1,Bcl-2,Bcl-2 associated X prorein(Bax),Cleaved-cysteinyl aspartate specific proteinase-3(Cleaved caspase-3),Beclin1,LC3,and P62 proteins in the cells in various groups.Results:Compared with control group,t

关 键 词：腹主动脉瘤 人血管平滑肌细胞 叉头框转录因子O1 自噬 细胞凋亡 

分 类 号：R732.2[医药卫生—肿瘤]