腺病毒介导的人ING4基因诱导C6鼠胶质瘤细胞凋亡

Cellular Apoptosis of C6 Mouse Glioma Cells Induced by hING4 Mediated by Adenovirus

目的:研究肿瘤抑制基因人ING4(inhibitor of growth family,member4)对C6鼠胶质瘤细胞的促凋亡作用。方法:将携有绿色荧光蛋白(GFP)腺病毒空载体Ad及重组腺病毒Ad-hING4-His(由本科室构建)分别感染C6细胞,RT-PCR法检测hING4的转录,Western-blotting法检测目的蛋白的表达。并观测hING4基因表达对C6胶质瘤细胞的作用,用MTT法绘制生长曲线,计算抑瘤率。再取重组腺病毒Ad-hING4-His及空腺病毒Ad作用后的C6细胞分别行激光共聚焦显微镜观察凋亡小体、透射电镜观察亚细胞结构的变化,抽提基因组DNA行琼脂糖凝胶电泳及流式细胞仪检测。结果:Ad-hING4-His感染C6细胞后,RT-PCR及Western-blotting结果提示有目的基因的转录和表达。hING4基因表达可以显著抑制C6细胞生长。激光共聚焦观察可见明显核断裂、透射电镜可见实验组细胞呈凋亡表现、基因组DNA电泳呈现梯形条带,流式细胞仪检测有明显AP峰,凋亡率达18.1%。结论:hING4可以通过促进细胞凋亡作用而显著抑制C6细胞的增殖和生长。

The known members of inhibitor of growth （ING） gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to regulate brain tumour angiogenesis through transcriptional repression of NF-κB-responsive genes, induce G2/M arrest by the increased p21 expression in a p53-dependent manner, suppress the loss of contact inhibition and represses activation of the hypoxia inducible factor, which plays an important role in the progression of tumorigenesis. However, seldom studies about ING4 inducing tumor cells apoptosis were reported. The C6 cells （ mouse glioma cells） were infected respectively with the blank adenovirus carrying GFP （Ad） and the recombinated Ad-hING4-His, then RT-PCR assay was used to detect the transcriptions of hING4, as well Western-blotting assay was ued to detect the expressions of hING4. The effects of hING4 expression upon C6 cells were observed, and the growth curve was drawed and tumor control rates were calculated. The C6 cells, which were affected by blank Ad and Ad-hING4- His, were respectively observed by LSCM （laser scan confocal microscope） and transmission electron microscope （TEM）, detected by flow cytometry; and the genomic DNA of both groups were extracted and electrophoresised in agarose gel to examinate the DNA fragments. The results showed hING4 can significantly inhibit the growth of C6 cells by promoting the cell＇ s apoptosis, which probably is the first one to prove this property of ING4. The experimental and theoretical foundation for gene therapy for gliomas with ING4 in the future was established.