Electrotransfection by human telomerase reverse transcriptase gene:optimization in precartilagious stem cell culture / 中国组织工程研究

ABSTRACT

BACKGROUND:The human telomerase reverse transcriptase gene (hTERT) transfected into target cel s can play an important role in target cel proliferation and differentiation by increasing telomerase activity and maintaining telomere length. OBJECTIVE:To explore the effect of hTERT transfection on telomerase activity and biological characteristics of precartilage stem cel s culured in vitro. METHODS:Precartilage stem cel s cultured in vitro were subjected to hTERT gene transfection via a retrovirus vector pLXSN. Meanwhile, control and negative control groups were set up. After transfection, TRAP-ELISA assay was used to detect telomerase activity;RT-PCR and western blot employed to detect hTERT mRNA and protein expressions;cel counting kit-8 used to detect cel proliferaiton based on cel growth curve;and flow cytometry adopted to detect cel cycle and distribution. RESULTS AND CONCLUSION:The telomerase activity was significantly increased at 48 hours after hTERT gene was transfected into the precartilage stem cel s. After transfection of hTERT, hTERT mRNA and protein levels were significantly increased, the cel growth rate was significantly increased, the proportion of cel s at G 0/G 1 phase was decreased, and the number of S-phased cel s increased compared with the control group and negative control group. There were significant differences among the groups (P<0.05). In conclusion, hTERT transfection via retrovirus vector pLXSN can promote the proliferation of precartilage stem cel s in rats by increasing the telomerase activity.