Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (49): 9189-.doi: 10.3969/j.issn.1673-8225.2010.49.016

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Expression of recombinant coexpression plasmid of pIRES-hVEGF121cDNA/hBMP-4 in bone marrow mesenchymal stem cells and its effects on osteogenic differentiation

Zhou Zhi-lin1, Sun Jun1, Zhao Yu2, Wang Chun-lan2   

  1. 1Department of Orthopaedics, Anhui Provincial Children’s Hospital, Pediatric Clinical College, Anhui Medical University, Hefei  230051, Anhui Province, China; 2Department of Plastic Surgery, First Affiliated Hospital, Anhui Medical University, Hefei  230022, Anhui Province, China
  • Online:2010-12-03 Published:2010-12-03
  • Contact: Sun Jun, Master, Professor, Chief physician, Department of Orthopaedics, Anhui Provincial Children’s Hospital, Pediatric Clinical College, Anhui Medical University, Hefei 230051, Anhui Province, China
  • About author:Zhou Zhi-lin★, Master, Physician, Department of Orthopaedics, Anhui Provincial Children’s Hospital, Pediatric Clinical College, Anhui Medical University, Hefei 230051, Anhui Province, China andyzlin@sina.com
  • Supported by:

    the Natural Science Foundation of Anhui Province, No. 070413090*

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Abstract:

BACKGROUND: Under some conditions, human body tissue repair is the result of common coordination of many factors. Thus, the construction of coexpression of many genetic carriers can meet the requirement of gene therapy of multiple factors, and is the new idea of recent gene therapy.
OBJECTIVE: To observe the expression of recombinant coexpression plasmid of pIRES-hVEGF121cDNA/hBMP-4 in transfected rat bone marrow mesenchymal stem cells (BMSCs) and its effects on the differentiation of rat BMSCs into osteoblasts, and provide a basis for combination gene therapy of bone defects. 
METHODS: BMSCs were isolated and cultivated from the bone marrow of rats by the whole bone marrow culture method. BMSCs were analyzed by the flow cytometry to detect the surface antigens. Recombinant coexpression plasmids of pIRES-hVEGF121cDNA/hBMP-4 were confirmed by restriction enzymolysis and then transfected rat BMSCs by lipofectamine. The total RNA and total proteins extracted from the resulting combination were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. Alizarin red staining was utilized to detect the differentiation of rat BMSCs after transfection.
RESULTS AND CONCLUSION: Flow cytometry analysis showed that BMSCs were strong positive for CD90, but did not express CD45. Recombinant coexpression plasmid of pIRES-hVEGF121cDNA/hBMP-4 was transfected successfully and the plasmid could coexpress hVEGF121cDNA and hBMP-4 efficiently in rat BMSCs. Alizarin red staining presented strong positive for 14 days after transfection. Human VEGF121 and BMP-4 gene can induce rat BMSCs to differentiate into osteoblasts, which provides a basis for combined gene therapy of bone defects.

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