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目的构建重组逆转录病毒载体pMSCV-ANGPTL4,并检测该重组逆转录病毒载体所介导的人ANGPTL4基因的抗肿瘤作用。方法将ANGPTIA cDNA亚克隆至逆转录病毒载体质粒 pMSCV。用脂质体法将重组病毒pMSCV-ANGPTL4转染HepG2细胞。流式细胞术和荧光显微镜检测 HepG2细胞绿色荧光蛋白的表达。RT-PCR法检测ANGPTL4 mRNA的表达。检测HepG2细胞体内外生长情况。结果重组病毒pMSCV-ANGPTL4和空病毒pMSCV的病毒滴度分别为1．4×106和 1．5×106感染性病毒颗粒／ml。表达GFP的HepG2-ANGPTL4细胞组和HepG2-pMSCV细胞组的阳性细胞率分别为68．45％和77．72％。HepG2-ANGPTL4细胞组和HepG2-pMSCV细胞组的平均荧光强度比对照组HepG2细胞分别高31．68倍和64．87倍。HepG2-ANGPTL4细胞组ANGPTL4 mRNA的表达分别是HepG2-pMSCV细胞组和HepG2细胞组的154％和161％。HepG2-ANGPTL4细胞组的体外增殖速度较之HepG2-pMSCV细胞组和HepG2细胞组明显降低(P<0．01)。HepG2-ANGPTL4细胞荷瘤裸鼠肿瘤的平均体积和重量均较HepG2-pMSCV细胞组和HepG2细胞组明显降低(P<0．01)。结论获得稳定转染ANGPTL4基因的人肝癌细胞株HepG2-ANGPTL4。重组逆转录病毒载体所介导的人 ANGPTL4基因转移很可能是一种有效的肝癌基因治疗方式。
Objective To construct recombinant retroviral vector pMSCV-ANGPTL4 and test the antitumor effect of human recombinant ANGPTL4 mediated by the recombinant retroviral vector. Methods ANGPTIA cDNA was subcloned into retroviral vector plasmid pMSCV. Recombinant virus pMSCV-ANGPTL4 was transfected into HepG2 cells by liposome method. Flow cytometry and fluorescence microscopy were used to detect the expression of green fluorescent protein in HepG2 cells. The expression of ANGPTL4 mRNA was detected by RT-PCR. Detection of HepG2 cells in vitro and in vivo growth. Results The virus titers of the recombinant viruses pMSCV-ANGPTL4 and empty virus pMSCV were 1.4 × 10 6 and 1.5 × 10 6 infectious virus particles / ml, respectively. The positive rate of HepG2-ANGPTL4 cells and HepG2-pMSCV cells expressing GFP were 68.45% and 77.72% respectively. The average fluorescence intensity of HepG2-ANGPTL4 cells and HepG2-pMSCV cells were 31.68 times and 64.87 times higher than those of HepG2 cells. The ANGPTL4 mRNA expression in HepG2-ANGPTL4 cells was 154% and 161% of HepG2-pMSCV cells and HepG2 cells respectively. The proliferation rate of HepG2-ANGPTL4 cells was significantly lower than that of HepG2-pMSCV cells and HepG2 cells (P <0.01). The average tumor volume and weight of HepG2-ANGPTL4 tumor-bearing nude mice were significantly lower than those of HepG2-pMSCV and HepG2 cells (P <0.01). Conclusion The human hepatoma cell line HepG2-ANGPTL4 stably transfected with ANGPTL4 gene was obtained. Recombinant retroviral vector mediated human ANGPTL4 gene transfer may be an effective way of gene therapy of liver cancer.