外源hTERT基因对大鼠肝细胞增殖的影响
Effects of exogenous hTERT on proliferation of primary rat hepatocytes
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摘要: 目的: 构建含目的基因人端粒逆转录酶(hTERT)重组逆转录病毒载体pLNCX2-hTERT,体外感染原代大鼠肝细胞,观察对大鼠肝细胞增殖的影响。方法: 将hTERT插入逆转录病毒载体pLNCX2获得重组逆转录病毒载体pLNCX2-hTERT,转染包装细胞293T后可获得表达hTERT逆转录病毒,感染体外培养的原代大鼠肝细胞,通过RT-PCR及western-blot检测基因在细胞中的表达情况。结果: 重组载体pLNCX2-hTERT经双酶切鉴定及序列测定,证实构建正确。转染原代大鼠肝细胞后能明显促进其增殖。结论: 体外转染原代大鼠肝细胞后能有效促进其增殖,这为进一步解决人工肝细胞来源问题奠定了实验基础。Abstract: Objective: To construct a new type of retrovirus plasmid pLNCX2 hTERT and to investigate the effects of human telomerase reverse transcriptase(hTERT) gene delivery on primary cultured rat hepatocyte proliferation. Methods: The plasmid pLNCX2-hTERT was transformed into 293T cells to generate recombinant retrovirus.The expression of hTERT in primary cultured rat hepatocytes infected with pLNCX2-hTERT was detected by RT-PCR and western blot.The impact of hTERT on the proliferative activity of cells was examined by methyl thiazol tetrazolium(MTT). Results: The recombinant retrovirus plasmid pLNCX2-hTERT was correctly constructed.When the primary cultured rat hepatocytes were infected with the viruses,the growth of primarily-cultured cells can be significantly increased. Conclusion: The recombinant retrovirus plasmid pLNCX2-hTERT can transfer hTERT into hepatocytes and increase the cell proliferation significantly.
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