ING4及IL-24双基因共表达腺病毒载体对MG-63人骨肉瘤细胞体外抑癌增效作用研究

Growth-inhibition Effect of Recombinant Adenovirus Expressing ING4 and IL24 Genes on MG-63 Osteosarcoma Cells in Vitro

目的用人肿瘤生长抑制因子(mING4)及人白介素24(IL-24)基因构建腺病毒双基因共表达载体(Ad-ING4-IL-24),并设Ad-ING4和Ad-IL-24单基因对照,研究双基因共表达载体对MG-63人骨肉瘤细胞体外抑癌增效作用.方法首先将已构建成功的Ad-ING4-IL-24,Ad-ING4及Ad-IL-24腺病毒感染QBI-293A细胞,经多轮扩增后获得高滴度的病毒子,测病毒效价后分别以100 MOI剂量感染MG-63细胞,用荧光显微镜、RT-PCR法检测ING-4及IL-24在MG-63细胞中的转录和表达;MTT法检测ING-4及IL-24基因表达对MG-63细胞的生长抑制效应;半定量RT-PCR法检测ING4及IL-24基因表达对MG-63细胞中的Bcl-2,Bax,Caspase3,p21,CD34等相关基因表达的影响.结果获得了高滴度的重组腺病毒Ad-ING4-IL-24,Ad-ING4,Ad-IL-24和Ad-GFP;治疗后MG-63细胞中ING-4及IL-24双基因表达对MG-63细胞增殖有明显抑制作用,ING-4及IL-24基因表达可通过上调细胞中Bax,Caspase3,p21和下调Bcl-2,CD34基因表达诱导细胞凋亡.在病毒子剂量为100 MOI时,Ad-ING4-IL-24对MG-63人骨肉瘤细胞抑制作用比对Ad-ING4和Ad-IL-24抑制效果更为显著.结论腺病毒介导的ING4或/和IL-24基因可明显抑制MG-63人骨肉瘤细胞的生长,且Ad-ING4-IL-24双基因比Ad-ING4和Ad-IL-24单基因的抑制效果更为显著,该现象可能是通过改变Bcl-2,Bax,Caspase3,p21,CD34基因表达水平来发挥抗肿瘤作用的.

Objective By using the tumor growth inhibitory factor（mING4） and human interleukin 24（IL-24） gene of adenovirus gene co-expression vector（Ad-ING4-IL-24）,and a single gene Ad-ING4 and Ad-IL-24 single gene control,to study the synergistic inhibitory effect of the vector on human osteosarcoma MG-63 cells in vitro.Method The Ad-ING4-IL-24,Ad-ING4 and Ad-IL-24 adenovirus infected QBI-293A cells were constructed,and the high-titer virus was obtained after many rounds of the amplification;the viral titers were measured and MG-63 cells were infected with a dose of 100 MOI,and the transcription and the expression ING-4 and IL-24 in MG-63 cells were detected by fluorescence microscopy and RT-PCR assay;inhibitory effects of ING-4 and IL-24 gene expression on the proliferation of MG-63 cells were detected by MTT;Effects of the expressions of ING4 and IL-24 genes on Bcl-2,Bax,Caspase3,p21,CD34 and other related gene expressions in MG-63 cells were measured with semi-quantitative RT-PCR method.Results The recombinant adenoviruses,including Ad-ING4-IL-24,high titers of Ad-ING4,Ad-IL-24 and Ad-GFP,were obtained;after the treatment,the double-gene expressions of ING-4 and IL-24 in MG-63 cells showed a significantly inhibitory effects on the proliferation of MG-63 cells,the expression of ING-4 and IL-24 genes could induce the apoptosis of cells by up-regulating the expression of Bax,Caspase3 and p21 in the cells,and down-regulating Bcl-2 and CD34 gene expressions.At the dose of 100 MOI,the inhibitory effects of Ad-ING4-IL-24 on human osteosarcoma MG-63 cells were more significant than those of Ad-ING4 or Ad-IL-24.Conclusion The adenovirus mediated ING4 or/and IL-24 genes can inhibit the growth of human osteosarcoma cell line MG-63 significantly,and the inhibitory effects of Ad-ING4-IL-24 genes should be more significant than those of Ad-ING4 or Ad-IL-24 gene.This phenomenon indicates that the antitumor effect may be exserted by changing the expression levels of Bcl-2,Bax,Caspase3,p21 and CD34 genes.