hTERT基因转染激活人毛乳头细胞端粒酶的实验研究

Experimental study of hTERT transfection activating telomerase in human dermal papilla cells

目的:明确外源性人端粒酶反转录酶(hTERT)基因转染能否激活体外培养人毛乳头细胞(DPC)的端粒酶。方法:采用脂质体转染法,将空载体质粒pIRES2-EGFP和含有hTERT基因的质粒pIRES2-EGFP-hTERT分别导入体外培养的正常人DPC,经G418筛选得到阳性克隆,连续传代培养。应用反转录(RT)-PCR法检测hTERTmRNA的表达,端粒重复序列扩增(TRAP)-ELISA法检测细胞端粒酶活性。结果:未转染DPC和空载体转染细胞(DPC-EGFP)无hTERTmRNA表达,端粒酶活性为阴性;而外源性hTERT基因转染细胞(DPC-hTERT)稳定表达hTERTmRNA,同时端粒酶活性转为阳性。结论:外源性hTERT基因转染能够激活体外培养人DPC的端粒酶,为建立永生化细胞系奠定基础。

Objective： To explicate whether the telomerase can be activated by human telomerase reverse transcriptase （hTERT） in human dermal papilla cells （DPC）. Methods： The plasmid plRES2-EGFP and plasmid plRES2-EGFP-hTERT encoding hTERT were transfected into cultured normal human DPC, then the positive cells were selected with G418. After the positive cells were expanded in culture, the expression of hTERT mRNA was detected by RT-PCR, and the telomerase activity was detected by TRAP-ELISA. Results： The hTERT wasn＇t expressed at mRNA level and the telomerase activity was negative in untransfected DPC and vacant vector transfected cells（DPC-EGFP）. The hTERT was expressed at mRNA level and meanwhile the telomerase activity was positive in plRES2-EGFP-hTERT transfected cells（DPC-hTERT）. Conclusion： Telomerase can be activated by hTERT in human DPC. This is the first step to establish immortalized human DPC line.