腺病毒介导PSMA3基因过表达对肝癌SMMC7721细胞增殖、周期、凋亡及其荷瘤生长的影响  

Effects of adenovirus-mediated PSMA3 gene overexpression on proliferation,cell cycle and apoptosis of human hepatocellular carcinoma SMMC7721 cells

作　　者：南映瑜 项颖 龚奕 肖春燕 杨涛 张文军 黄德鸿 李启英 童永红 Nan Yingyu;Xiang Ying;Gong Yi;Xiao Chunyan;Yang Tao;Zhang Wenjun;Huang Dehong;Li Qiying;Tong Yonghong(Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment,Chongqing University Cancer Hospital&Chongqing Cancer Institute&Chongqing Cancer Hospital)

机构地区：[1]重庆大学附属肿瘤医院/重庆市肿瘤研究所/重庆市肿瘤医院肿瘤转移与个体化诊治转化研究重庆市重点实验室

出　　处：《重庆医科大学学报》2019年第12期1582-1588,共7页Journal of Chongqing Medical University

基　　金：重庆市科委应用开发计划资助项目(编号:CSTC2014yykfA110031);中国医师协会研究资助项目(编号:ZGYSXH20120701)

摘　　要：目的:构建蛋白酶体亚基α3(proteasome subunit alpha type 3,PSMA3)基因腺病毒重组载体,并观测腺病毒介导PSMA3基因过表达对肝癌SMMC7721细胞增殖、周期、凋亡及其荷瘤生长的影响。方法:RT-PCR法扩增PSMA3基因全长CDs序列后,克隆入pTG19-T载体,再亚克隆入pAdTrack-CMV腺病毒穿梭载体中,构建重组pAdTrack/PSMA3腺病毒载体,随后与骨架质粒pAdEasy-1同源重组形成p Ad/PSMA3重组腺病毒,经包装及滴度测定,获高感染力的pAd/PSMA3病毒。用pAd/PSMA3病毒感染人肝癌SMMC7721细胞,荧光显微镜观测被感染的SMMC7721细胞的绿色荧光蛋白(green fluorescent protein,GFP)的变化情况。Cell counting kit-8(CCK-8)法检测pAd/PSMA3病毒对SMMC7721细胞增殖的影响。Real-time PCR和Western blot分别检测被感染的SMMC7721细胞的PSMA3基因、增殖细胞核抗原基因(proliferating cell nuclear antigen,PCNA)、Caspase-3促凋亡基因及其编码蛋白的表达情况。流式细胞仪(FCM)检测被感染的SMMC7721的周期、凋亡变化。将经pAd/PSMA3病毒感染的SMMC7721细胞移植裸鼠皮下以生成荷瘤,逐日观察荷瘤的生长情况。所有实验以空病毒感染及未感染的细胞做对照。结果:克隆到768 bp的PSMA3基因,并构建了其具高感染力的pAd/PSMA3重组病毒。CCK-8法结果表明,过表达PSMA3基因的SMMC7721细胞,其增殖明显减慢(P<0.05)。Real-time PCR、Western blot检测显示,pAd/PSMA3重组病毒可介导SMMC7721细胞过表达PSMA3基因,上调其Caspase-3基因及其编码蛋白的表达,并下调其PCNA基因及其蛋白的表达(P<0.05)。FCM检测证实,过表达PSMA3基因的细胞可被阻滞于G1期(细胞周期),并可促使其凋亡,其凋亡率差异有统计学意义(P<0.05)。动物实验证实,p Ad/PSMA3病毒可明显抑制裸鼠荷瘤的生长(P<0.01)。结论:重组病毒pAd/PSMA3可将SMMC7721细胞阻滞于G1期(细胞周期),抑制其增殖,促使其凋亡,并可抑制其裸鼠荷瘤的生长,这些可能和下调其增殖相�Objective:To construct the adenovirus vector of proteasome subunit alpha type 3(PSMA3)gene and investigate whether the adenovirus vector carrying PSMA3 gene affecting the proliferation,cell cycle,apoptosis and the growth of nude mice bearing tumors of human hepatocellular carcinoma cell line SMMC7721.Methods:cDNA fragment encoding human PSMA3 gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR).The PCR amplified fragment was first cloned into pTG19-T vector,and then subcloned PSMA3 gene into the shuttle vector pAdTrack-CMV for constructing the recombinant plasmid pAdTrack/PSMA3,which homologously recombinated with the adenoviral backbone vectors Adeasy-1 to generate recombinant adenoviral plasmids Ad/PSMA3.The recombinant adenoviruses Ad/PSMA3 were generated by transfecting the recombinant adenoviral DNA into 293 cells and then employed to infect SMMC7721 cells.The expression of enhanced green fluorescent protein(EGFP)in transfected SMMC7721 cells were observed by fluorescence microscope.The proliferation of transfected SMMC7721 cells were tested via Cell count kit-8(CCK-8),and the expreesion levels of PSMA3,proliferating cell nuclear antigen(PCNA)and caspase-3 mRNAs proteins in transfected SMMC7721 cells were detected by RT-PCR and Western blot,respectively.The cell cycle distribution and apoptosis rate of transfected SMMC7721 cells were analyzed by flow cytometry(FCM).SMMC7721 cells infected with p Ad/PSMA3 virus were transplanted subcutaneously into nude mice to generate tumors,and the growth of the tumors was observed day by day.Results:PSMA3 gene was successfully cloned.Adenoviral virus particles which possess infectious competent were produced from Ad/PSMA3.The results of CCK-8 revealed that the proliferation of SMMC7721 cells,which overexpressed PSMA3,were slower than the control cells,especially from 48 h after transfection(P<0.01).The results of RT-PCR and Western blot demonstrated that SMMC7721 cells transfected with pAd/PSMA3 recombinant virus can effectively overexpressed PSMA

关 键 词：蛋白酶体亚基α3 SMMC7721细胞 增殖 凋亡 荷瘤 

分 类 号：R73.35.4[医药卫生—肿瘤]