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作 者:熊建军[1] 干丽君[1] 吴周环[1] 吴萍[1] 王亦东[1] 李卫东[1]
出 处:《中国现代医学杂志》2010年第8期1161-1163,1167,共4页China Journal of Modern Medicine
摘 要:目的构建高表达ARLTS1基因的逆转录病毒载体(PLEGFP-IRES-ARLTS1),探讨外源性ARLTS1基因对人肝癌细胞HepG2增殖的影响。方法 RT-PCR扩增ARLTS1 cDNA,克隆至重组逆转录病毒载体PLEGFP-IRES。重组质粒转染病毒包装细胞PT67,产生病毒后感染HepG2细胞,荧光显微镜检测绿色荧光蛋白表达,Western Blot检测细胞内ARLTS1蛋白表达,并对感染后细胞的增殖状况进行分析。结果酶切及基因测序证实重组质粒PLEGFP-IRES-ARLTS1构建成功;荧光显微镜观察到70%左右靶细胞内有绿色荧光蛋白表达;Western Blot证实,ARLTS1蛋白在HepG2-ARLTS1内表达显著升高;MTT实验显示,HepG-2-ARLTS1细胞组体外增殖率较各对照组明显降低(P<0.05)。结论获得了稳定表达外源性ARLTS1蛋白的HepG2细胞株,外源性ARLTS1抑制HepG2细胞增殖,为后续研究ARLTS1抗肿瘤机制奠定了基础。【Objective】To construct the retroviral vector expressing ARLTS1 gene(PLEGFP-IRES-ARLTS1) and to investigate the effect of exogenous ARLTS1 on proliferation in hepatoma carcinoma cell HepG2.【Methods】 ARLTS1 cDNA was amplified by RT-PCR and subcloned into recombinant retrovirus vector PLEGFP-IRES.Plasmids were transfected into retrovirus packaging cell PT67.The target cells HepG2 were infected with retroviral particles,fluorescence microscopy detected expression of GFP in HepG2 cells.The expression of ARLTS1 protein was determined by Western Blot.The proliferation of cells was analysised by MTT assay.【Results】Restrictive endonuclease identification and gene sequencing confirmed that the recombinant vector was successfully constructed.Fluorescence microscopy observed the expression of GFP in target cells.Western Blot demonstrated the level of ARLTS1 protein was obviously increased in HepG2-ARLTS1 cells.The proliferation rate of HepG2-ARLTS1 cells group was lower than control groups(P 0.05).【Conclusions】The HepG2 cells line for stable expressing ARLTS1 was established and expression of exogenous ARLTS1 decreased HepG2 cells viability.These results laid a foundation for further research the anti-tumor mechanism of ARLTS1.
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