pIRES-IL-24-TRAIL基因载体的构建及其对肝癌细胞迁移和凋亡的影响  

Construction of pIRES-IL-24-TRAIL vector and its influences on the motility and apoptosis of hepatocarcinoma cells

作　　者：魏杨辉[1] 黄耀[1] 刘娟 张卫星[1] 吴爱国 

机构地区：[1]深圳市第四人民医院普外科,广东深圳518033 [2]南方医科大学珠江医院普外科,广东广州510515

出　　处：《中国现代医学杂志》2014年第19期21-25,共5页China Journal of Modern Medicine

基　　金：2012年广东省医学科研基金(No:B2012330);2011年深圳市科技计划项目(No:201103165);2010年深圳市福田区科技计划项目(No:2010FTWS06)

摘　　要：目的构建真核表达载体pIRES-IL-24-TRAIL,探讨其对肝癌细胞迁移和凋亡的影响。方法以人胎盘组织总RNA为模板,采用RT-PCR二步法,扩增TRAIL的cDNA序列,将其克隆入pIRES载体,扩增IL-24的cDNA序列,将其克隆入pIRES-TRAIL载体,构建其真核瞬时表达载体pIRES-IL-24-TRAIL,以Lipofectamine2000转染技术,将TRAIL、IL-24基因导入肝癌细胞Bel-7402和PLC,流式细胞仪检测各组细胞凋亡情况,划痕实验比较不同组间细胞迁移情况。结果①克隆到TRAIL、IL-24基因的cDNA序列,成功构建其真核表达载体;②流式细胞仪发现,实验组比对照组凋亡率高(P<0.05);③细胞划痕实验显示实验组细胞迁移能力明显低于对照组(P<0.05)。结论 pIRES-IL-24-TRAIL构建成功,并能诱导肝癌细胞凋亡、降低其迁移能力。【Objective】To construct the eukaryotic expression vector pIRES-IL-24-TRAIL and investigate its influences on the motility and apoptosis of hepatocarcinoma.【Methods】cDNA fragment encoding human TRAIL gene wasamplified by reverse transcriptase-polymerase chain reaction（RT-PCR） with the total mRNA isolated from human placenta tissue as template. The PCR amplified fragment of TRAIL gene was cloned into pIRES vector. And then, cDNA fragment encoding human IL-24 gene was amplified. The PCR amplified fragment of IL-24 gene was subcloned into the recombinant eukaryotic expression vector pIRES-TRAIL to construct pIRES-IL-24-TRAIL plasmid after sequencing. Subsequently,plasmid DNA of pIRES- IL-24- TRAIL was transfected into hepatocarcinoma cell Bel-7402 and PLC with the help of Lipofectamine2000 transfection reagent. Cell apoptosis was measured with flow cytometer. Wound healing test was performed for cell motility assay. 【Results】①cDNA sequence encoding human TRAIL and IL-24 gene was successfully cloned,and recombinant eukaryotic expression vector pIRES-IL-24-TRAIL was constructed.②The apoptosis rate by flow cytometer was higher in the transfected group than the control group（P〈 0.001）. ③Wound-healing assay shows that cell motility could be more effectively suppressed in the transfected group than the control group（P〈 0.001）.【Conclusion】pIRES-IL-24-TRAIL was constructed successfully, and it can promote the apoptosis of hepatocarcinoma cell and impede tumor cell motility.

关 键 词：白介素-24 肿瘤坏死因子可溶性相关凋亡诱导配体 肝癌 迁移 凋亡 

分 类 号：R735.7[医药卫生—肿瘤]