Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection  

作　　者：李克强 李文林 彭淑牖 石小玉 唐洪林 刘颖斌 

机构地区：[1]Department of General Surgery, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, China [2]Jiangxi Key Laboratory of Medical Biotechnology, Jiangxi Medical Science Institute, Nanchang 330006, China [3]Basic Medical Department of Jiangxi Medical college, Nanchang 330006, China

出　　处：《Chinese Medical Journal》2004年第9期1364-1369,共6页中华医学杂志（英文版）

摘　　要：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 lBackground 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 l

关 键 词：angiopoietin-like 4 · HepG 2 cell  · retroviral vector 

分 类 号：R73-3[医药卫生—肿瘤]