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目的:研究慢病毒介导人端粒酶逆转录酶(hTERT)基因对人肝细胞L02生物学特性的影响。方法:将表达hTERT基因的重组慢病毒感染人肝细胞L02,通过杀稻瘟筛选获得阳性克隆。分别采用实时荧光RT-PCR和PCR-ELISA方法检测转染前后L02肝细胞的hTERT-mRNA水平和端粒酶活性的变化。通过MTT法、流式细胞术(FCM)及RT-PCR等方法观察其形态和生物学功能的变化。结果:转染后的L02肝细胞存在hTERT基因过表达和端粒酶活性上调,体外培养条件下维持肝细胞原有形态,存活期显著延长,增殖能力较强,同样具有表达和分泌ALB及糖原合成的功能。结论:外源性hTERT基因的异位表达不仅上调L02肝细胞的端粒酶活性,而且促进其增殖而不影响基本功能。
Objective: To study the effect of lentivirus-mediated human telomerase reverse transcriptase (hTERT) gene on the biological characteristics of human hepatocyte L02. Methods: Recombinant lentivirus expressing human telomerase reverse transcriptase (hTERT) gene was transfected into human hepatocyte L02 and positive clones were screened by blast killer blast. Real-time fluorescence RT-PCR and PCR-ELISA were used to detect the changes of hTERT-mRNA and telomerase activity in L02 hepatocytes before and after transfection. Morphological and biological changes were observed by MTT assay, flow cytometry (FCM) and RT-PCR. Results: The transfection of L02 hepatocytes had hTERT gene overexpression and telomerase activity up-regulated. The original morphology of hepatocytes was maintained under in vitro culture conditions, the survival period was significantly prolonged, the proliferative capacity was strong, and also the expression and secretion of ALB and sugar The original synthesis function. Conclusion: Ectopic expression of exogenous hTERT gene not only up-regulated telomerase activity of L02 hepatocytes, but also promoted its proliferation without affecting the basic function.