Research For The Tet-on Lentiviral Vectors Carrying HBMP-2 Transfecting Human Umbilical Cord Mesenchymal Stem Cells And Expression Of HBMP-2 In Vitro

Objective To research the condition of Tetracycline-regulated lentiviral vector containing hBMP-2 transfecting human umbilical cord mesenchymal stem cells (HUCMSCs), and to study the inducible expression of hBMP-2 in HUCMSCs.Methods HUCMSCs were cultured according to literature methods. In the day before transfection, the third or fourth generation HUCMSCs with well growth state were inoculated into six-well plates with 40% confluents. In the trasfection day, pLV/EXPN2-Puro-EF1A-rtTA, pLV/EXPN2-Neo-TRE-hBMP2 were transfected into HUCMSCs together, then change the culture medium after 8 hours. Puromycin and G418 were added into the culture medium for stable screening. DNA was extracted of from transfected HUCMSCs and checked by PCR in order to determine whether the gene was successfully transferred. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxcycline concentrations in the same induction time, and the other by the same concentration in different time. The expression of target gene hBMP-2 was identified by ELISA KIT. After2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Alizarin Bordeaux staining method.Results Tet-on-regulated lentiviral vector containing hBMP-2 could infect HUMSCs with high efficiency. The expression of hBMP-2 reached the peak at 10μg/ml Doxcycline at 48 hours of induction. After 2-week osteogenic induction, a lot of mineralization nodes were observed.Conclusion Tet-on-regulated lentiviral vector containing hBMP-2 could infect HUMSCs with high efficiency successfully, and the inducible expression of hBMP-2 was researched, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.