CAJ | 학술논문

目的探讨外源性人端粒酶反转录酶(hTERT)基因转染对人胎儿表皮干细胞增殖的影响.方法采用脂质体转染法,将含有hTERT基因的质粒pIRES2-EGFP-hTERT和卒载体质粒pIRES2-EGFP分别导人体外培养的人胎儿表皮干细胞,并用G418筛选法进行抗性克隆的筛选.用RT-PCR和Western blot检测表皮十细胞hTERT mRNA及其蛋白的表达,端粒重复序列扩增法(TRAP)-ELISA检测其端粒酶活性,流式细胞仪检测细胞周期.结果非转染组和空载体质粒转染组表皮干细胞弱表达hTERT mRNA和蛋白,而质粒转染组表皮干细胞强表达hTERT mRNA和蛋白;与非转染组和窄载体质粒转染组比较,质粒转染组表皮干细胞端粒酶活性的表达量显著升高,G_0/G_1期细胞比例明显下降,而S、G_2/M期细胞比例升高,增殖指数增加.结论外源性hTERT基因转染能有效增强体外培养的人表皮干细胞hTERT mRNA和蛋白的表达及端粒酶活性,细胞增殖能力明显增强.
목적 탐토외원성인단립매반전록매(hTERT)기인전염대인태인표피간세포증식적영향.방법 채용지질체전염법,장함유hTERT기인적질립pIRES2-EGFP-hTERT화졸재체질립pIRES2-EGFP분별도인체외배양적인태인표피간세포,병용G418사선법진행항성극륭적사선.용RT-PCR화Western blot검측표피십세포hTERT mRNA급기단백적표체,단립중복서렬확증법(TRAP)-ELISA검측기단립매활성,류식세포의검측세포주기.결과 비전염조화공재체질립전염조표피간세포약표체hTERT mRNA화단백,이질립전염조표피간세포강표체hTERT mRNA화단백;여비전염조화착재체질립전염조비교,질립전염조표피간세포단립매활성적표체량현저승고,G_0/G_1기세포비례명현하강,이S、G_2/M기세포비례승고,증식지수증가.결론 외원성hTERT기인전염능유효증강체외배양적인표피간세포hTERT mRNA화단백적표체급단립매활성,세포증식능력명현증강.
Objective To investigate the changes of human epidermal stem cells after transfected with human telomerase reverse transcriptase(hTERT)gene.Methods The plasmid pIRES2-EGFP and plasmid pIRES2-EGFP-hTERT encoding hTERT were transfected into in vitro cultured human fetal epidermal stem cells by liposome-mediated transfection.Then,the positive cells were selected with G418.The mRNA and protein expressions of hTERT were detected by reserve transcriptase-polymerase chain reaction(RT-PCR)and Western blot.The telomerase activity and the proliferation and cycle of human epidermal stem cells were detected by telomeric repeat amplification protocol(TRAP)-ELISA and flow cytometry respectively.Results RT-PCR and Western blot techniques detected weak mRNA and protein expressions of hTERT gene in untransfected and vacant vector transfected cells but high level of mRNA and protein expressions of hTERT gene in pIRES2-EGFP-hTERT transfected cells.Compared with untransfected and vacant vector transfected cells,the pIRES2-EGFP-hTERT transfected cells had higher telomerase activity,with lower proportion of cells at G_0/G_1 phase,higher proportion of cells at S and G_2/M phases and enhanced proliferation ability.Conclusion Transfection with hTERT gene can markedly enhance mRNA and protein expressions,telomerase activity and proliferation ability of hTERT gene of human epidermal stem cells euhured in vitro.