摘要
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 biological behavior changes and the possible mechanisms of human osteosarcoma cell line U-2OS after hTERT gene modifications. Methods Transform hTERT gene to U-2OS cells (hTERT negative) by liposome method. Then test the expres- sion of hTERT by inmmnochemistry and Western blot. Detect the changes of telomere length, telomerase activity, cell cycle of U-2OS, biological and vito-dynamic changes and transwell matrigel ability after hTERT gene modification. Results lmmunohistochemical staining and Western blot showed that the expression of hTERT was positive in U-2OS cells after hTERT gene modification. The telomerase activity was upregulated, and the telomere was extended. The vacuum micropipette aspiration assay found that hTERT transfection increased the cellular adhesion capacity to the extra- cellular matrix(ECM). Transwell matrigel assay confirmed an increased invasion ability in hTERT/ U2OS cells. Conclusion These results strongly suggest that hTERT transfection promotes the in- vasion of telomerase-negative cells. Telomerase-mediated telomere maintenance enables these cells to achieve a fully malignant endpoint, including invasion and metastasis.
出处
《实用临床医药杂志》
CAS
2010年第3期13-20,共8页
Journal of Clinical Medicine in Practice
基金
国家自然科学基金资助项目(30871150)
重庆市杰出青年科学基金资助项目(CSTC
2009BA5045)
关键词
端粒酶逆转录酶
端粒延长替代途径(ALT)
侵袭
相对端粒长度
转移
Human telomerase reverse transcriptase
alternative lengthening of telomeres (ALT)
invasion
relative telomere length
metastasis