胰岛素样生长因子结合蛋白7表达质粒的构建及其瞬时转染对SMMC7721肝癌细胞增殖的影响  

Construction of p IRES2-Zs Green1-IGFBP7 and effect of IGFBP7 overexpression on survival ability of SMMC7721 cells

作　　者：岳春燕[1] 彭健[1] 胥洪鹃[1] 杨满意[1] 

机构地区：[1]中南大学湘雅医院,湖南长沙410008

出　　处：《中国现代医学杂志》2015年第12期11-15,共5页China Journal of Modern Medicine

基　　金：中南大学研究生自主探索创新项目(No:2013zzts307)

摘　　要：目的采用TA克隆的方法构建IGFBP7表达载体,考察IGFBP7过表达对SMMC7721细胞生存活力的影响。方法 RT-PCR法调取IGFBP7表达基因,构建p MD19-T-Simple-IGFBP7载体,测序鉴定后与p IRES2-Zs Green1质粒进行拼接构建表达质粒;随后表达质粒转染SMMC7721细胞,RTFQ-PCR测定SMMC7721细胞中IGFBP7 m RNA水平,MTT检测IGFBP7表达质粒转染对SMMC7721细胞增殖的影响。结果 IGFBP7-c-DNA设计长度为840 bp,电泳结果可在750~1 000 bp观察到清晰单一条带,表明成功调取IGFBP7基因;pMD19-TS-IGFBP7载体和p-IRES2-Zs-Green1-IGFBP7表达质粒测序结果正确,表明表达质粒构建成功;RTFQ-PCR结果显示IGFBP7转染组细胞IGFBP7 m RNA水平显著升高;MTT结果显示IGFBP7转染组细胞增殖明显下降。结论成功用TA克隆的方法构建了IGFBP7表达载体,IGFBP7的过表达能显著抑制SMMC7721细胞增殖。[Objective]To construct a vector expressing insulin-like growth factor binding protein 7（IGFBP7）using TA cloning and investigate the effect of IGFBP7 overexpression on SMMC7721 cells. [Methods]IGFBP7c DNA was obtained by RT-PCR and ligated to p MD19-T Simple vector. Then sequencing was performed. After double digestion, IGFBP7 c DNA was linked to p IRES2-Zs Green1 plasmid. p IRES2-Zs Green1-IGFBP7 was transfected into SMMC7721 cells with Lipofectmine2000. Real-time fluorescent quantitative PCR（RTFQ-PCR） was used to quantify the expression of IGFBP7. MTT was performed to evaluate the effect of IGFBP7 on proliferation and apoptosis of SMMC7721 cells. [Results]PIRES2-Zs Green1-IGFBP7 was constructed successfully. RTFQ-PCR results showed that IGFBP7 expression significantly increased in the p IRES2-Zs Green1-IGFBP7 group. Proliferation of p IRES2-Zs Green1-IGFBP7 group was significantly inhibited compared with the control group. [Conclusions]IGFBP7 expression vector has been successfully constructed by TA cloning with a high efficiency. And IGFBP7 overexpression could significantly inhibit proliferation of SMMC7721 cells.

关 键 词：TA克隆 胰岛素样生长因子结合蛋白7 实时荧光定量PCR 增殖 

分 类 号：R73-3[医药卫生—肿瘤]