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目的观察hTERT基因修饰对人骨肉瘤细胞系U-2OS生物学行为的影响。方法采用脂质体法将克隆有人全长cDNAhTERT的真核荧光质粒(pIRES2-EGFP-hTERT)转染端粒酶阴性的人骨肉瘤U-2OS细胞,经G418筛选,免疫组化和Westorn Blot鉴定后,检测其端粒酶活性的改变、生长周期以及生物力学的变化。结果成功转染人真核荧光表达载体的U-2OS细胞能有效抵抗G418,并可有效表达hTERT蛋白,细胞内端粒酶活性明显增强,G1期细胞比例下降,S期细胞比例升高,并且还明显增强了对细胞外基质的粘附力。进一步采用Transwell小孔迁移实验检测发现,hTERT/U2OS侵袭能力明显增强。结论转染hTERT基因后,U-2OS细胞内可同时通过端粒酶途径延长端粒。hTERT基因修饰可促进细胞周期进程,促使细胞从G1期→S期。通过替代途径延长端粒的肿瘤细胞在hTERT基因修饰后,增殖能力及侵袭能力明显增强。
Objective To investigate the effect of hTERT gene modification on the biological behavior of human osteosarcoma cell line U-2OS. Methods The human telomerase reverse transcripts of human osteosarcoma U-2OS cells were transfected with pIRES2-EGFP-hTERT, which was cloned by full-length cDNA hTERT, and identified by G418, immunohistochemistry and Westorn Blot The changes of telomerase activity, growth cycle and biomechanics were detected. Results U-2OS cells successfully transfected with human eukaryotic expression vector were effective against G418, hTERT protein was effectively expressed, the telomerase activity was significantly increased, the proportion of cells in G1 phase decreased and the proportion of S phase cells increased, and It also significantly enhances the adhesion to the extracellular matrix. Further experiments using Transwell pinhole migration assay showed that the invasion ability of hTERT / U2OS was significantly enhanced. Conclusion The hTERT gene transfected U-2OS cells can telomere telomerase extension. hTERT gene modification can promote cell cycle progression and promote cells from G1 phase to S phase. Proliferation of telomere tumor cells by alternative means of hTERT gene modification, proliferation and invasion ability was significantly enhanced.