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目的利用凋亡素和IL18基因的抗肿瘤优势,设计构建共表达凋亡素和hIL-18基因的真核重组子,检测其在体外对人肝癌细胞BEL7402的作用效果,旨在探索新的肿瘤基因治疗方法。方法将凋亡素VP3基因与hIL-18基因一同克隆到真核表达载体pVAX1上,构建重组质粒pVVP3IL-18,经脂质体介导,转染人肝癌细胞BEL7402,AO/EB染色,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)法和流式细胞仪分别分析凋亡细胞的形态学变化、DNA断裂情况和不同细胞周期的DNA含量变化,透射电镜分析凋亡细胞超微结构变化。结果随着转染时间的延长,重组质粒pVVP3IL18所表达的目的蛋白对BEL7402细胞的杀伤作用逐渐增强,作用48h,形态学观察表明BEL7402细胞发生明显的凋亡,凋亡率可达(56.5±11.9)%,并且大部分细胞为TUNEL阳性着色,流式细胞术分析可见明显的亚二倍体凋亡峰,透射电镜观察可见细胞核固缩、染色质聚集等典型的凋亡超微结构变化。结论联合应用凋亡素与hIL18基因,在体外对人肝癌细胞BEL7402具有明显的凋亡诱导作用。
OBJECTIVE: To construct eukaryotic recombinants expressing apoptin and hIL-18 gene by using the anti-tumor advantage of apoptin and IL18 gene, and to detect their effect on human hepatocellular carcinoma cell BEL7402 in vitro, so as to explore new tumor Gene therapy methods. Methods VP3 gene and hIL-18 gene were cloned into eukaryotic expression vector pVAX1 to construct recombinant plasmid pVVP3IL-18. The recombinant plasmid pVVP3IL-18 was transfected into human hepatocellular carcinoma cell line BEL7402 by Lipofectamine 2000. AO / EB staining, Nucleotide transferase-mediated dUTP nick end labeling (TUNEL) and flow cytometry were used to analyze morphological changes of apoptotic cells, DNA fragmentation and changes of DNA content in different cell cycle. Transmission electron microscopy was used to analyze apoptotic cells Microstructure changes. Results With the extension of transfection time, the killing effect of the target protein expressed by recombinant plasmid pVVP3IL18 on BEL7402 cells gradually increased. The morphological observation showed that the apoptosis of BEL7402 cells was obvious (56.5 ± 11.9 )%, And most of the cells were TUNEL-positive staining. Flow cytometry analysis showed obvious sub-diploid apoptotic peak. Transmission electron microscopy showed typical apoptotic ultrastructure changes such as nuclear condensation and chromatin aggregation. Conclusion Combined application of apoptin and hIL18 gene has obvious apoptosis-inducing effect on human hepatocellular carcinoma cell BEL7402 in vitro.