Krüppel-like factor 4慢病毒表达载体构建及其对胃癌细胞BGC-823生物学行为的影响  

Construction of Krüppel-like factor 4 lentiviral vector and its impact on biological behaviors of gastric cancer cells BGC-823

作　　者：张能[1] 张军[1] 王子卫[1] 査郎 何苗[1] 

机构地区：[1]重庆医科大学附属第一医院胃肠外科,重庆400016

出　　处：《中国老年学杂志》2012年第24期5445-5448,共4页Chinese Journal of Gerontology

基　　金：重庆市卫生局医学科学研究重点项目(2010-1-19)

摘　　要：目的构建Krüppel-like factor4(KLF4)过表达慢病毒载体,探讨其对胃癌细胞株BGC-823生物学行为的影响。方法检测BGC-823中KLF4 mRNA的表达水平;用真核表达质粒pcDNA3.1IE-KLF4-EGFP,将KLF4基因连入慢病毒载体pLv-UbC-IRES2-EGFP中,构建pLv-KLF4-IRES2-EGFP重组慢病毒表达载体。将酶切和测序鉴定后的重组质粒转染至BGC-823中,观察转染情况,RT-PCR检测KLF4 mRNA。慢病毒包装后转染BGC-823细胞,Western印迹检测KLF4蛋白。结果重组质粒经酶切和DNA测序证实目的基因插入正确;pcDNA3.1IE-KLF4-EGFP转染BGC-823细胞后KLF4 mRNA升高。慢病毒包装后转染BGC-823检测到目的蛋白KLF4。KLF4能够将细胞阻滞于G1/S,抑制其生长、促进细胞凋亡,减少细胞侵袭能力。结论转染BGC-823后KLF4蛋白检测证实慢病毒载体成功构建,KLF4能抑制胃癌细胞的恶性转化。Objective To construct the lentiviral vectors of Krüppel-like factor 4 （KLF4） and investigate its impact on the biological behaviors of human gastric cancer cell lines BGC-823. Methods KLF4 mRNA expression levels were detected in gastric cancer cell lines BGC-823, KLF4 gene was imported into lentiviral vector pLv-UbC-IRES2-EGFP by eukaryotic expression plasmid pcDNA3. 11E-KLF4- EGFP for constructing recombinant lentiviral pLv-KLF4-IRES2-EGFP expression vector. Recombinant plasmid was transfected into gastric cancer cell lines BGC-823 after restriction enzyme digestion and sequencing. Details of transfection were observed and KLF4 mRNA was detected by RT-PCR. KLF4 protein was measured by Western blot in gastric cancer cell lines BGC-823 which were transfected by packaged lentiviral vector. Results The correct gene insertion in recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing, KLF4 mRNA increased after pcDNA3. 11E-KLF4-EGFP was transfected into gastric cancer cells BGC-823. Target protein KLF4 was detected in gastric cancer cells BGC-823 that were transfected by packaged lentiviral vectors. KLF4 blocked the cell cycle at G1/S, inhibited their growth, promoted apoptosis and reduced cell invasion. Conclusions Successful construction of KLF4 lentiviral vectors is confirmed by KLF4 proteins expression in BGC-823 cells that are transfected, KLF4 can inhibit malignant transformation of gastric cancer cells.

关 键 词：Krüppel-like FACTOR 4(KLF4) 慢病毒载体 构建及验证 胃癌细胞 

分 类 号：R735.2[医药卫生—肿瘤] Q812[医药卫生—临床医学]