科研之友
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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 antitumor 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 x 10(6) 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, 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.
