摘要
目的:构建重组人源化hING4基因并探究其对肺癌细胞的生长抑制效应。方法:以pcDNA3.0-mING4重组质粒为模板,通过定点突变技术构建带有绿色荧光蛋白(GFP)的重组腺病毒Ad-hING4基因。用荧光显微镜和RT-PCR法检测hING4在肺癌细胞中的表达;Western印迹法检测hING4对肺癌细胞中Bax、Bcl-2表达的影响;集落形成试验和FCM法检测hING4对肺癌细胞增殖和凋亡影响。结果:基因测序和PCR结果提示成功构建人源化Ad-hING4基因;并能在肺癌细胞中表达,对肺癌细胞有细胞毒作用;Western印迹法检测结果提示基因在上调Bax表达同时下调Bcl-2的表达;集落形成试验、FCM检测结果提示hING4基因可抑制肺癌细胞增殖并诱导凋亡。结论:成功构建了Ad-hING4基因,并能在体外抑制肺癌细胞生长。
Objective ：To construct a recombinant human inhibitor of growth 4 （hING4） gene and observe its growth inhibition effect on lung adenocarcinoma NCI H460 cells. Methods： The GFP-labeled recombinant adenovirus vector hING4 （ Ad-hING4 ） was constructed using pcDNA3.0-mING4 plasmid as a template and site-specific mutagenesis technique. RT-PCR and fluorescence microscope were used to detect the expression of hING4 in NCI H460 cells. Bax and Bcl-2 protein expression were detected by Western blotting. Colony formation assay and flow cytometry were used to observe the effect of hING4 on the proliferation and apoptosis of H460 cells. Results： DNA sequence analysis and PCR indicated that Ad-hING4- were successfully constructed. Ad-hING4 was expressed in lung cancer H460 cells and had obvious cytopathic effect （CPE）. Western blotting suggested that hING4 gene caused up-regulation of Bax expression and down-regulation of Bcl-2 expression. Colony formation assay and flow cytometry showed that hING4 inhibited the proliferation of H460 cells and induced apoptosis of lung cancer cells. Conclusion：Ad-hING4 gene was successfully constructed. It inhibited the growth of lung cancer H460 cells in vitro.
出处
《肿瘤》
CAS
CSCD
北大核心
2008年第11期942-945,共4页
Tumor
关键词
肺肿瘤
ING4蛋白
人
诱变
突点
基因
肿瘤抑制
Lung neoplasms
ING4 protein, human
Mutagenesis, site-directed
Genes, tumor suppressor
