慢病毒介导的FHIT基因过表达调控人肝癌细胞株生长实验研究  

Effects of FHIT Overexpression Mediated by Slow Virus on the growth of Hepatoma Lines in Vitro

作　　者：戈佳云[1] 杨筱燕[2] 李晓[1] 吴雪松[3] 施智甜 赵松凌[1] 张文瀚[1] 王琳[1] 魏东[1] GE Jia-yun YANG Xiao-yan LI Xiao WU Xue-song SHI Zhi-tian ZHAO Song-ling ZHANG Wen-han WEI Dong(The 2rid Dept. of Hepato-biliary Surgergy Dept. of Hemodialysis Dept. of Gastrointenstinal Surgery, The 2nd Affiliated Hospital of Kunming Medical University, Kunming Yunnan 650101, China)

机构地区：[1]昆明医科大学第二附属医院肝胆外二科,云南昆明650101 [2]昆明医科大学第二附属医院血液透析科,云南昆明650101 [3]昆明医科大学第二附属医院胃肠外科,云南昆明650101

出　　处：《昆明医科大学学报》2016年第8期19-23,共5页Journal of Kunming Medical University

基　　金：国家自然科学基金资助项目(81061434)

摘　　要：目的探讨过表达FHIT基因对FHIT基因含量不一致的人肝癌细胞株Hep G2、Hep3B和Huh7的增殖、凋亡和细胞侵袭以及细胞周期的影响,以期证实FHIT基因在肝癌细胞株的表达差异与肝癌细胞的分化有密切相关性.方法体外成功构建p LVX-IRES-FHIT慢病毒重组载体转染FHIT基因含量不一致的肝癌细胞株Hep G2、Hep3B和Huh7,以MTT法、FCM法、Annexin V/PI双染色法、细胞周期检测技术和Transwell侵袭实验分析过表达的FHIT基因对三系肝癌细胞的增殖活性、凋亡、细胞侵袭性和细胞周期的影响机制及其生物学效应与时间梯度的关系.结果体外成功构建重组慢病毒载体p LVX-IRES-FHIT转染FHIT含量不一致的肝癌细胞株Hep G2、Hep3B和Huh7.(1)MTT法检测显示三系肝癌细胞株的p LVX-IRES-FHIT组细胞增殖活性明显降低,且随时间推移细胞增殖抑制效果更为显著,在48 h达到高峰,此后到72 h进入平台期,对照组细胞增殖抑制不明显(P<0.05).Annexin V/PI双染色法检测发现p LVX-IRES-FHIT组三系肝癌细胞48 h凋亡率较对照组差异无统计学意义(P>0.05).三系肝癌细胞中对p LVX-IRES-FHIT组转染致FHIT基因过表达抑制增殖活性大小的顺序依次是Hep G2>Hep3B>Huh7;(2)经过Transwell侵袭实验表明对FHIT基因过表达对三系肝癌细胞的p LVX-IRES-FHIT组侵袭性均有明显的抑制作用(P<0.05),而对照组未见明显差异(P>0.05).三系肝癌细胞的侵袭性顺序依次为Hep G2>Hep3B>Huh7;(3)细胞周期检测发现p LVX-IRES-FHIT组G0/G1期细胞增多,S期细胞减少,增殖指数减低,在Hep G2细胞中与对照组间有统计学意义(P<0.05),在Hep B和Huh7细胞中与对照组间无统计学意义(P>0.05).FHIT基因的过表达对三系肝癌细胞的细胞周期影响为细胞周期停滞于S期,且G0/G1期细胞增多的顺序依次是Hep G2<Hep3B<Huh7.结论 FHIT基因在肝癌细胞株中的表达差异可能与肝癌细胞的恶性分化有密切正相关性,FHIT基因有可能成为原Objective To observe overexpressional effects of FHIT gene on proliferation, apoptosis,invasion and cell cycle of human hepatocellular carcinoma cells including Hep G2,Hep3 B and Huh7. Me thod The p LVX- IRES- FHIT slow virus recombinant vector was constructed and transfected into Hep G2, Hep3 B and Huh7 cells. We detected the infection of cell proliferation, apotosis, invasion and cell cycle of these cells with overexpression of FHIT and analyzed the relationship of biological effects and time gradient by MTT,Annexin V/PI using cell cycle detection technology and Transwell experiment. Re s ults According to DNA sequencing, the p LVX- IRES- FHIT slow virus recombinant vector was constructed successfully.（1） MTT tests showed that proliferation activity of Hep G2,Hep3 B and Huh7 with p LVX- IRES- FHIT transfection was reduced significantly in48 hours and the inhibition was stable after 72 hours. The inhabitation in the control group was not significant（P〈0.05）. Annexin V/PI double staining method showed that no significant difference of apoptosis level was seen after48 h among three hepatoma cells（P 〉0.05）. The order of inhibitional proliferation activity from high to low was Hep G2, Hep3 B and Huh7;（2） Transwell tests showed that invasive activity of Hep G2, Hep3 B and Huh7 with p LVX- IRES- FHIT transfection was inhibited（P〈0.05）. The control group was not sinificantly inhibited（P〈0.05）. The order of invasive activity was Hep G2〉Hep3B〉Huh7;（3） Cell cycle test showed that the number of cells in G0/G1 stage increased whereas decreased in stage S. Hep3 B and Huh7 cells in the control group were not significantly inhibited（P〉0.05）. The influence of FHIT over expression to these cells was that cell cycle stopped in S stage, and sequence of cell number in G0/G1 was Hep G2 〈Hep3B 〈Huh7.Conclus ion A positive correlation was found between FHIT expression and cellular differentiation on hepatoma cells. FHIT gene may be a potential gene therapy for human hepatocellular carc

关 键 词：FHIT 慢病毒载体 增殖 凋亡 细胞侵袭性 细胞周期 

分 类 号：R735.7[医药卫生—肿瘤]