腺病毒载体介导的ING4基因对MG-63人骨肉瘤细胞的抑制作用  

The growth-inhibitory effect of recombinant ING4-Adenovirus on MG-63 osteosarcoma cells

作　　者：井莹莹[1] 杨吉成[1] 缪竞诚[1] 盛伟华[1] 胡志清[1] 单云波[1] 刘铁连[1] 韩亚丽[1] 包婉蓉[1] 

机构地区：[1]苏州大学医学部细胞与分子生物学教研室,苏州215123

出　　处：《中国免疫学杂志》2009年第3期234-239,共6页Chinese Journal of Immunology

基　　金：苏州大学医学发展基金资助项目(No.EE134517)

摘　　要：目的:用人源化改造的鼠肿瘤生长抑制因子(mING4)基因构建的腺病毒表达载体(Ad-ING4)研究对MG-63人骨肉瘤细胞的抑制作用。方法:以pcDNA3.0-mING-4重组质粒为模板,通过基因定点突变技术对mING4基因进行人源化改造,采用腺病毒载体的基因重组和体外包装技术获得表达与人ING-4氨基酸序列相同的重组腺病毒子(Ad-ING4),感染MG-63细胞,用荧光显微镜、RT-PCR法检测ING4在MG-63细胞中的转录和表达;MTT法和流式细胞技术检测ING4基因表达对MG-63细胞的生长抑制和凋亡效应;荧光共聚焦显微镜观察MG-63细胞凋亡的形态学变化;半定量RT-PCR法检测ING4基因表达对MG-63细胞中的Bax、Bcl-2基因表达的影响。结果:基因测序和PCR分析结果显示,人源化改造的小鼠ING4分子氨基酸序列与人ING4分子完全相同,成功构建了Ad-ING4腺病毒表达载体,在MG-63细胞中ING4基因的表达对MG-63细胞增殖有明显抑制作用,并可诱导细胞凋亡,出现典型细胞凋亡形态学变化,ING4基因表达可通过上调细胞中Bax和下调Bcl-2基因表达诱导细胞凋亡。结论:转染ING4基因可明显抑制MG-63人骨肉瘤细胞的生长,诱导细胞凋亡,该现象可能是通过改变Bax,Bcl-2表达水平来发挥抗肿瘤作用的。Objective：To construct recombinant adenovirus harboring humanarized mouse ING4 gene and to study its growth-inhibitory effects on osteosarcoma cells.Methods：In order to obtain the recombinant ING4 Adenovirus vector,site-specific mutagenesis technique was used,and pcDNA3.0-mING-4 was used as the template.Then the recombinant Ad-ING4 was obtained by gene recombination and in vitro packaging technique.Osteosarcoma cells were infected with Ad-ING4 and RT-PCR was used to detected the transcription of ING4 in the cells.Cell growth inhibition was detected by MTT assay.Morphologic changes were observed by light microscope and fluorescence microscope.The cell cycle and apoptosis were detected by flow cytometry,and morphologic changes of the infected cells were observed under laser scanning confocal microscopy（LSCM）.In addition,RT-PCR was used to detected the transcription of Bax and Bcl-2.Results：Ad-hING4 were proved in correct construction by gene sequence and PCR results.Expression of hING4 was identified by RT-PCR.By microscope and flow cytometry displayed the apoptosis of the osteosarcoma cells positively expressing ING4.The apoptotic body in the cells was shown by laser scanning confocal microscopy（LSCM）.Additionally,the mRNA of Bax was up-regulated and mRNA of Bcl-2 was down-regulated in the cells by RT-PCR.Conclusion：Ad-ING4 can inhibit the growth of MG-63 cells and induce cell apoptosis,which may result in changing Bax and Bcl-2 expression.

关 键 词：ING4基因 腺病毒载体 MG-63人骨肉瘤细胞 凋亡 

分 类 号：R392.11[医药卫生—免疫学]