Proliferation changes of human epidermal stem cells in response to transfection with human telomerase reverse transcriptase gene / 中华创伤杂志

ABSTRACT

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.