Background This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of reco...Background This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.Methods ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG 2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG 2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG 2-ANGPTL4 cells in vivo and in vitro, respectively.Results The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4×106 infective viral grains/ml, and the rate of HepG 2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG 2-ANGPTL4 cells than in HepG 2 cells. The expression of ANGPTL4 mRNA in HepG 2-ANGPTL4 cells was higher than in HepG 2-pMSCV cells (154% higher) or HepG 2 cells (161% higher). The proliferation rate of HepG 2-ANGPTL4 cells in vitro was obviously lower than those of HepG 2-pMSCV cells and HepG 2 cells (P<0.01). The mean volume and weight of tumors seeded from HepG 2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG 2 cells and HepG 2-pMSCV cells (P<0.01).Conclusion A stable ANGPTL4-transfected human liver cancer cell line HepG 2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy.展开更多
文摘Background This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.Methods ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG 2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG 2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG 2-ANGPTL4 cells in vivo and in vitro, respectively.Results The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4×106 infective viral grains/ml, and the rate of HepG 2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG 2-ANGPTL4 cells than in HepG 2 cells. The expression of ANGPTL4 mRNA in HepG 2-ANGPTL4 cells was higher than in HepG 2-pMSCV cells (154% higher) or HepG 2 cells (161% higher). The proliferation rate of HepG 2-ANGPTL4 cells in vitro was obviously lower than those of HepG 2-pMSCV cells and HepG 2 cells (P<0.01). The mean volume and weight of tumors seeded from HepG 2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG 2 cells and HepG 2-pMSCV cells (P<0.01).Conclusion A stable ANGPTL4-transfected human liver cancer cell line HepG 2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy.
