论文部分内容阅读
目的研究高表达ABCG2基因对人原发性肝癌细胞SP表型的影响。方法构建pMSCVpuro-ABCG2逆转录病毒质粒,并转染至病毒包装细胞PT67中,将获得的病毒上清液感染人原发性肝癌non-SP(Side population)细胞,筛选稳定感染的细胞系non-SP-ABCG2和non-SP-puro,RT-PCR法检测细胞中ABCG2基因的转录水平,Western blot法检测细胞中ABCG2蛋白的表达水平,显微镜观察细胞形态的变化,Hoechst33342染色分析细胞的SP表型。结果逆转录病毒质粒pMSCVpuro-ABCG2经双酶切及测序证实构建正确;non-SP-ABCG2细胞中ABCG2基因的转录和蛋白表达水平均较空载体pMSCVpuro转染的细胞non-SP-puro明显提高;non-SP-ABCG2细胞的形态发生改变,成梭形生长,偶见小三角样细胞,细胞生长缓慢,细胞间排列不紧密,并出现了SP表型。结论 ABCG2基因的高表达与细胞SP表型的形成具有一定的相关性,并能影响细胞的基本形态。
Objective To study the effect of high expression of ABCG2 gene on the phenotype of SP in human primary hepatoma cells. Methods The retroviral vector pMSCVpuro-ABCG2 was constructed and transfected into the PT67 virus-infected cell line. The obtained virus supernatant was transfected into human primary hepatocellular carcinoma (NSC) non-SP cells and the stable infected cell lines were selected. ABCG2 and non-SP-puro were used to detect the expression of ABCG2 in cells. The expression of ABCG2 protein was detected by Western blot. The morphology of cells was observed by microscope. The SP phenotype was analyzed by Hoechst33342 staining. . Results The correct construction of retrovirus plasmid pMSCVpuro-ABCG2 was confirmed by double enzyme digestion and sequencing. The expression of ABCG2 gene in non-SP-ABCG2 cells was significantly increased compared with the non-SP-puro cells transfected with empty vector pMSCVpuro. The morphology of non-SP-ABCG2 cells changed into fusiform growth, occasionally small triangular-like cells, slow cell growth, cell arrangement is not close, and the SP phenotype. Conclusion The high expression of ABCG2 gene has certain correlation with the formation of SP phenotype and can affect the basic morphology of cells.