Construction Immortalized Human Bone Mesenchymal Stem Cells

Objective: Construction immortalized human Bone Mesenchymal stem cells.Methods: HBMSCs cells were infected by the retroviral particles(pBabepuro- hTERT), and then the designed hTERT-hBMSCs cells were selected by puromycin. The telomerase activity, the cells growth curve and Chromatosome kayotype of hTERT-hBMSCs cells were detected by TRAP (PCR)-ELISA and MTT respectively. Then, the hTERT-hBMSCs cells were induced into osteoblast by NH OsteoDiff Medium and the ALP was detected by immunocytechemical staining in hTERT- hBMSCs cells. Finally, proliferation capacity of hTERT-hBMSCs cells were observed by the rate of colony formation.Results: hBMSCs cells were attached to the surface of flasks with spindle-shaped or fibrocyte-like shape. The phenotype of hTERT-hBMSCs was CD44 positive and CD34 negative. The telomerase activity of hTERT-hBMSCs cells was positive. The growth speed of hTERT-hBMSCs cells were higher than those of hBMSCs cells measured by MTT assay .The growth speed of hBMSCs cells were slower than those of hTERT-hBMSCs cells under the same conditions. Presently it had propagated eleven generations, but they didn't exhibit senescent phenomenon. The analysis of chromatosome of hTERT-hBMSCs cells showed that chromatosome were nomal diploid .The hTERT-hBMSCs cells maintained their differentiational capability which proved by induced to osteoblast in vitro. The rate of hTERT-hBMSCs cells colony formation was lower than that of Hela cells. In a word, all the results showed that the characteristics of hBMSCs cells infected by the retroviral particles(pBabepuro -hTERT)were not changed.Conclusion: The hTERT gene activates the telomerase and hTERT-hBMSCs keep some biological characteristics of hBMSCs cells.