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目的:以pBABE-tert重组反转录病毒为载体,介导端粒酶反转录酶基因(hTRT)转染体外培养的人口腔角化细胞,探讨端粒酶对细胞寿命的影响。方法:将含有hTRT的pBABE-tert重组反转录病毒载体扩增后,转染体外培养的人口腔角化细胞,挑选阳性克隆进行传代培养,以PCR-ELISA和PCR-PAGE电泳检测端粒酶活性。结果:获得端粒酶活性稳定表达的口腔角化上皮细胞克隆,寿命可延长至8~9代。结论:转导端粒酶反转录酶基因的人口腔角化细胞寿命延长1倍,但不能永生化,说明人口腔上皮角化细胞的永生化是复杂和多点调控的过程,端粒酶活性增加是细胞永生化的关键但不是唯一原因。
OBJECTIVE: To investigate the effect of telomerase on the cell lifespan in vitro using pBABE-tert recombinant retrovirus as a vector to transduce telomerase reverse transcriptase gene (hTRT) into human oral keratinocytes. Methods: The hTERT-containing pBABE-tert recombinant retroviral vector was amplified and transfected into human oral keratinocytes cultured in vitro. The positive clones were selected for subculturing and the telomerase activity was detected by PCR-ELISA and PCR-PAGE active. Results: The oral keratinocyte epithelial cell clone with stable expression of telomerase activity could be extended to 8-9 generations. CONCLUSION: The population of human keratinocytes transduced with telomerase reverse transcriptase gene is doubled in lifespan, but it can not immortalize, indicating that the immortalization of human oral keratinocytes is a complex and multi-point process. Telomerase Increased activity is the key to immortalization but not the only reason.