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目的观察8型腺相关病毒介导的TRAIL基因对结肠癌HCT116细胞凋亡的诱导作用并探讨其作用机制。方法采用三质粒磷酸钙共沉淀方法转染HEK293细胞,制备出携带TRAIL 基因的8型重组腺相关病毒;重组病毒感染结肠癌细胞及正常人肝细胞,利用台盼蓝染色计数法测定细胞生长活性;光镜下观察病毒感染后细胞大体形态改变;Hoechst染色荧光显微镜下观察细胞核形态的改变;DNA凝胶电泳观察细胞凋亡的发生;蛋白印迹检测凋亡相关基因caspase-3,caspase-8蛋白表达的变化。结果携带TRAIL基因的8型重组腺相关病毒能够激活caspase通路,促进结肠癌细胞凋亡,有效抑制结肠癌细胞的生长,而对正常人肝细胞的生长没有影响。结论 8型腺相关病毒介导的TRAIL基因能够抑制结肠癌细胞HCT116的体外生长,对正常细胞没有毒性。
Objective To observe the effect of type 8 adeno-associated virus-mediated TRAIL gene on the apoptosis of colon cancer HCT116 cells and explore its mechanism of action. METHODS: Three-plasmid calcium phosphate co-precipitation method was used to transfect HEK293 cells. Type 8 recombinant adeno-associated virus carrying TRAIL gene was prepared. The recombinant virus was used to infect colon cancer cells and normal human hepatocytes. The activity of cell growth was determined by trypan blue staining. The morphology of the cells was observed under light microscope. Morphological changes of the nucleus were observed under fluorescence microscope using Hoechst staining. Apoptosis was observed by DNA gel electrophoresis. The caspase-3 and caspase-8 proteins were detected by Western blot. Change in expression. Results The recombinant adeno-associated virus type 8 carrying TRAIL gene can activate the caspase pathway, promote the apoptosis of colon cancer cells, effectively inhibit the growth of colon cancer cells, but has no effect on the growth of normal human hepatocytes. Conclusion Type 8 adeno-associated virus-mediated TRAIL gene can inhibit the growth of colon cancer cell HCT116 in vitro and has no toxicity to normal cells.