Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (36): 6673-6678.doi: 10.3969/j.issn.1673-8225.2011.36.006

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Osteogenous differentiation of bone marrow mesenchymal stem cells transfected with bone morphogenetic protein 4 and bone morphogenetic proteins 4/7

Yuan Shao-hui, Liu Wei, Wu Bin-qi, Han Xi-guang, Bi Zheng-gang   

  1. Department of Orthopedics, the First Affiliated Hospital of Harbin Medical University, Harbin  150001, Heilongjiang Province, China
  • Received:2011-03-02 Revised:2011-05-30 Online:2011-09-03 Published:2011-09-03
  • Contact: Bi Zheng-gang, Chief physician, Professor, Department of Orthopedics, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China
  • About author:Yuan Shao-hui☆, Doctor, Associate chief physician, Department of Orthopedics, the First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China yuansh1970@sina.com
  • Supported by:

    the Natural Science Foundation of Heilongjiang Province, No. D200876*; the Science and Technology Research Project of Heilongjiang Education Bureau, No. 11531149*

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Abstract:

BACKGROUND: Heterodimer of bone morphogenetic protein (BMP) has higher activity than BMP homodimer. Currently, there are no reports addressing comparison between BMP-4/7 heterodimer and BMP4 homodimer to induce osteogenic activity.
OBJECTIVE: To compare the difference in osteogenous differentiation of bone marrow mesenchymal stem cells (BMSCs) transfected with BMP4 and BMP-4/7, and to investigate biology activity of BMP-4/7 fusion gene.
METHODS: The mature peptide of BMP-4 and BMP-7 were gained by one-step RT-PCR from the human palcenta, and the BMP-4/7 fusion gene was gained through gene recombinant techniques and then transferred to pGEM plasmid. The BMP4 gene and BMP-4/7 fusion gene was cut down from the pGEM plasmid and the recombination was successfully completed in colibacillus and recombinant adeno-assosiated was produced in 293 cells. The BMSCs cell lines which could express each of the target protein w ere selected out by G418 and RT-PCR were used to certificate the expression of exogenous gene in BMSCs. Rabbit BMSCs were transfected with the recombinant adeno-assosiated virus vectors carrying BMP4 and BMP-4/7 fusion gene and cell reproductive activity detected by MTT method. The ossification of cells was evaluated by investigating the shape change of the cell ability of alkaline phosphatase and osteocalcin after transfection for 7 days.
RESULTS AND CONCLUSION: We successfully constructed the recombinant adeno-assosiated virus with BMP-4 gene and BMP-4/7 fusion gene; RT-PCR results certificated the confirm expression of the exogenous gene in BMSCs. The transfection efficiency of AAV-BMP4 and AV-BMP-4/7 were 68.20% and 72.18%. MTT showed that almost all of the BMSCs which had been transfected with exogenous gene had more strong proliferative potential than the untransfected group. The cell multiplication of BMP-4 fusion gene was stronger than BMP4. There was significantly higher alkaline phosphatase and osteocalcin in AAV-BMP4 and AAV-BMP-4/7 transfection groups than untransfection group. BMP-4/7 group had a stronger osteogenous differentiation than the BMP4 group (P < 0.01). The BMP4 and AAV-BMP-4/7 fusion gene can transfect rabbit BMSCs cultured in vitro at high transfection rate, and have significant ossification of activity. The BMP-4/7 fusion gene has a stronger osteogenous differentiation than the BMP4 single gene.

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