摘要
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 a eukaryotie recombinant plasmid for coexpression of apoptin and hIL-18 genes,determine its effect on human liver cancer BEL-7402 cells in vitro and provide a basis for exploring a new gene therapeutics for tumor. Methods Clone chicken anemia virus VP3 gene and human interleukin-18 （hIL-18 ） gene into eukaryotie expression vector pVAX1 and transfect the recombinant plasmid pVVP3IL-18 to human liver cancer BEL-7402 cells in the mediation of liposome. Observe the morphological change, DNA breakage, DNA content at different stages of cell cycle and ultrastructure of the apoptotic cells by AO/EB staining,termi- nal deoxynucleotidyl transferase-mediated dUTP nick end labeling（TUNEL） assay ,flow cytometry and transmission electron microscopy respectively. Results Along with the increasing hours after transfeetion, the killing effect of expressed product of recombinant plasmid pVVP3IL-18 on BEL-7402 cells was enhanced gradually. The morphological apoptosis of cells was observed 48 h after transfeetion. The apoptotic rate reached（56. 5±11.9） % and the majority of cells were TUNEL positive. Typical apoptotic sub-G1 peak was observed by flow eytometry. Typical ultrastructure change of apoptotic cells, such as pyknosis and ehromatin condensation, were observed by transmission electron microscopy. Conclusion The combined application of apoptin and hlL-18 genes induced the apoptosis of human liver cancer BEL-7402 cells in vitro significandy.
出处
《中国生物制品学杂志》
CAS
CSCD
2006年第3期229-232,共4页
Chinese Journal of Biologicals
基金
国家"973"资助项目(G1999011902).
