【摘要】 目的:探讨ANGPTL4基因及其缺失突变体对肝癌细胞SMMC-7721生长的影响。方法:构建ANGPTL4基因全长及其功能结构域缺失突变体的融合表达载体,即ANGPTL4-GFP-N1、ANGPTL4-del1-GFP-N1、ANGPTL4-del2-GFP-N1、ANGPTL4-del3-GFP-N1,脂质体法将EGFP-N1空载体、ANGPTL4基因及其缺失突变体分别转染肝癌细胞SMMC-7721,G418筛选,建立稳定细胞系,用MTT试验法、细胞集落形成试验检测细胞体外生长活性和增殖能力;流式细胞仪检测ANGPTL4基因对细胞凋亡的影响;裸鼠移植瘤试验检测转染细胞的体内成瘤特性。结果:ANGPTL4基因及其缺失突变体体外对肝癌细胞SMMC-7721的生长、集落形成具有明显的抑制作用(均为P<0.01);ANGPTL4全长基因及其缺失突变体ANGPTL4-del1-GFP-N1和ANGPTL4-del2-GFP-N1均对SMMC-7721细胞体内成瘤有明显抑制作用(均为P<0.01)。但转染ANGPTL4全长基因及其缺失突变体的SMMC-7721细胞的细胞凋亡发生率与转染空载体组相比没有明显差别(均为P>0.05)。结论:ANGPTL4基因及其缺失突变体对肝癌SMMC-7721细胞体内、外生长均具有明显抑制作用,但该抑制作用不是通过促进细胞凋亡实现的。更多还原

【Abstract】 Objective:To explore the effect of angiopoietinlike 4(ANGPTL4) gene and its deletion mutants on growth of hepatocellular carcinoma SMMC-7721 cells.Methods:ANGPTL4 ORF cDNA and its deletion mutants in functional domains(fibronectin like domain,signal peptide,coiled-coil domain) were cloned into EGFP-N1 vector and named as ANGPTL4-GFP-N1,ANGPTL4-del1-GFP-N1,ANGPTL4-del2-GFP-N1,and ANGPTL4-del3-GFP-N1,respectively.Hepatocellular carcinoma SMMC-7721 cells were transfected with EGFP-N1 empty vector as control and ANGPTL4-GFP-N1,ANGPTL4-del1-GFP-N1,ANGPTL4-del2-GFP-N1,and ANGPTL4-del3-GFP-N1 by lipofectAMINE mediation.The stable colonies were obtained by G418 screening.The growth of SMMC-7721 cells were studied by in vitro colony formation assay and MTT assay.The ectopic tumor formation was assessed in tumor-bearing nude mice.Apoptosis and cell cycle of SMMC-7721 cells were analyzed by FACS.Results:Compared with control,transfection of ANGPTL4 and its deletion mutants significantly inhibited the growth and colony formation of SMMC-7721 cells in vitro(P<0.01).Transfection of ANGPTL4 and its deletion mutants ANGPTL4-del1-GFP-N1 and ANGPTL4-del2-GFP-N1 also markedly inhibited the ectopic tumor formation of SMMC-7721 cells in nude Balb/c mice(all P<0.01).However,no significant difference in cell cycle and apoptotic ratio was observed between SMMC-7721 cells transfected with ANGPTL4/deletion mutants and those transfected with empty vector(P>0.05,P>0.05).Conclusion:(ANGPTL4) and its deletion mutants can inhibit growth of SMMC-7721 cells in vitro and in vivo.Non apoptosis-mediated mechanisms are thought to contribute to the inhibitory effects.更多还原