ZHOU Ming 1 , SHI Xinyan 2 , TIAN Shaoqi 1 , WANG Li 3 , ZHANG Jihua 4 , SUN Kang 1 , XING Shichao 5 , SUN Weixue 1 , HUANG Hongjie 1 , WU Dan 6
  • 1Joint Surgery, 2Medical Materials Office, 3Chinese-American Research Center for Stem Cells and Regenerative Medicine, 4Health Physical Examination Center, 5Central Laboratory, 6Plastic Surgery, the Affiliated Hospital of Medical College, Qingdao University, Qingdao Shandong, 266003, P.R.China.;
Inducible lentiviral vector; Human bone morphogenetic protein 2; Tet-on system; Human umbil ical cord blood mesenchymal stem cells; Doxycl ine
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Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results The
recombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.

Citation： ZHOU Ming,SHI Xinyan,TIAN Shaoqi,WANG Li,ZHANG Jihua,SUN Kang,XING Shichao,SUN Weixue,HUANG Hongjie,WU Dan. CONSTRUCTION OF INDUCIBLE LENTIVIRAL VECTOR CONTAINING HUMAN BONE MORPHOGENETIC PROTEIN 2 GENE AND ITS EXPRESSION IN HUMAN UMBILICAL CORD BLOOD MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2011, 25(4): 482-487. doi: Copy