摘要
目的探讨Akt2/Bad信号通路在非小细胞肺癌(NSCLC)凋亡中的作用机制。方法构建稳定过表达Bad同时稳定干扰Akt2的双病毒载体,将未感染慢病毒载体的H1299、H292、SPC-A1、H460及SK-MES-1细胞为NSCLC组,阴性对照病毒转染NSCLC细胞为NC组;稳定干扰Akt2的NSCLC细胞为shAkt2组;稳定过表达Bad的NSCLC细胞为Bad组;稳定干扰Akt2表达的同时稳定过表达Bad基因的NSCLC细胞为shAkt2+Bad组。通过体外实验,观察共感染组细胞凋亡及侵袭能力的变化。应用Western-blot技术检测各组NSCLC细胞株Bad、Akt2以及凋亡相关信号蛋白BCL-2、BCL-XL、caspase-9、Cyto-c蛋白的表达水平。体内构建H1299及SPC-A1荷瘤裸鼠模型,观测瘤体重量,利用TUNEL染色比较Akt2/Bad信号通路对肺癌细胞凋亡的作用。结果与NSCLC组比较,shAkt2+Bad组细胞和移植瘤体中Akt2蛋白水平明显减少(P<0.05),Bad蛋白水平显著增加(P<0.05)。shAkt2+Bad组细胞凋亡率明显高于shAkt2组及Bad组(P<0.05),H1299/SPC-A1体内荷瘤实验表明,shAkt2+Bad组瘤体重量明显低于Bad组、NSCLC组及NC组(均P<0.05),细胞凋亡率均高于其他3组(均P<0.05)。shAkt2和shAkt2+Bad组caspase-9表达显著降低;Bad组、shAkt2组和shAkt2+Bad组cyto-C表达显著增加,而BCL-2、BCL-XL在各组细胞中表达比较差异无统计学意义(P>0.05)。结论Akt2-Bad信号通路参与NSCLC的凋亡。
Objective To construct co-infected Bad overexpression and shAkt2 lentiviral vector systems NSCLC cell lines and explore the molecular crosstalk of Akt2 and BAD in NSCLC.Methods Bad over-expression lentivirus expressing vector infected shAkt2 NSCLC cells.After selection of Puromycin resistance cells,protein expression levels of Bad were determined western blot,respectively.The cells were divided into NSCLC normal control groups,Bad overexpression cells,shAkt2 cells,Bad+shAkt2 cells and their negative control(NC).Cells were characterized in vivo using apoptosis analysis and cell invasion assay.Expression levels of caspase-9,cyto-c,BCL-2 and BCL-XLwere determined by western-blot.Tumorigenicities of H1299 and SPC-A1 were assessed using nude mouse xenograft models in vivo.Results After co-infection of Bad+shAkt2 lentiviral vector systems in NSCLC cells,apoptosis were more significantly promoted than that of control and single lentiviral group in vitro and vivo(P<0.05).Bad+shAkt2 and shAkt2 groups showed lower caspase-9 expression(P<0.05).BAD,Bad+shAkt2 and shAkt2 groups showed higher cyto-c expression levels.Conclusion Silencing Akt2 combined with Bad overexpression have synergistic effect in promoting NSCLC cell apoptosis,inhibition of NSCLC cells tumorigenic ability.Targeted silencing of Akt2 combined with Bad overexpression can significantly increase the expression of cyto-c,down-regulating the expression of caspase-9,Akt2/Bad Signaling Passway could be involved in the molecular mechanim of NSCLC via promoting apoptosis.
作者
蒋莉
何芳
刘丹
李为民
陈渤江
JIANG Li;HE Fang;LIU Dan;LI Weimin;CHEN Bojiang(Department of Respiratory and Critical Care Medicine, The Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, Sichuan, China;Nanchong Central Hospital, The Second Clinical Medical College of North Sichuan Medical College, Nanchong 637000, Sichuan, China;North Sichuan Medical College, Nanchong 637000, Sichuan, China;Department of Respiratory and Critical Care Medicine, West China Hospital, Sichuan university, Chengdu 610041, China)
出处
《西部医学》
2021年第3期357-362,共6页
Medical Journal of West China
基金
国家自然科学基金重大研究计划(91859203)
四川省科技厅计划项目(2018JY0416)
南充市市校合作项目(18SXHZ0470)。