Study of hTERT activating telomerase in human bone marrow mesenchyme stem cells / 中国矫形外科杂志

ABSTRACT

[Objective]To explicate whether the telomerase activity is regulated by human telomerase reverse transcriptase (hTERT) in human bone marrow mesenchyme stem cells(hBMSC).[Method]hBMSC were cultured and transfected with eukaryotic expressing plasmid pCIneo-hTERT encoding hTERT. After selection with G418 to stabilize the transfection,expression of hTERT mRNA was detected with TR-PCR, detecting the expression of hTERT protein was detected with Western Bolt, and the telomerase activity in untransfected and transfected cells were detected by RT-PCR.[Result]The hMSCs grew well after transfecting plasmid pCIneo-hTERT.The cells began to suspend and die after the day of the G418 selection. At the tenth day,all the untransfected cells were dead, but the transfected cells began to clone proliferation. So the density of G418 subdued to 100 ?g/ml for maintaining selecting, at the twentieth day,there were obvious anti-G418 cell clones. After stable transfection, hTERT was expressed at mRNA and protein level in these anti-418 cell clones, and meanwhile telomerase activity was positive and obviously raise up in these transfected cells.[Conclusion]In human' bone marrow mesenchyme cells,telomerase could be activated by exogenous hTERT. This is a foundation to establish immortalized human bone marrow mesenchyme stem cell line.