LAI Jianming 1 , LIN Jianhua 2 , LIN Wenping 3 , WU Chaoyang 2
  • 1Department of Orthopedics, Longyan First Hospital Affiliated to Fujian Medical University, Longyan Fujian, 364000, P.R.China;;
  • 2Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University;;
  • 3Department of Orthopedics, the Second Affiliated Hospital of Fujian Medical University.;
Tissue engineering; Bone marrow mesenchymal stem cells; Transforming growth factor β1; Collagen type II; Articular cartilage; Gene therapy; Lentiviral vector; Pig
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Objective To construct recombinant lentiviral expression vectors of porcine transforming growth factor β1 (TGF-β1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-β1 gene-modified BMSCs for bone and cartilage tissue engineering. Methods The TGF-β1 cDNA was extracted and packed into lentiviral vector, and positive clones were identified by PCR and gene sequencing, then the virus titer was determined. BMSCs were isolated from
bone marrow of the 2-month-old Bama miniature pigs (weighing 15 kg), and the 2nd and 3rd generations of BMSCs were
harvested for experiments. BMSCs were then transfected by TGF-β1 recombinant lentiviral vectors (TGF-β1 vector group)
respectively at multi pl icity of infection (MOI) of 10, 50, 70, 100, and 150; then the effects of transfection were detected by
laser confocal microscope and Western blot was used to determine the optimal value of MOI. BMSCs transfected by empty vector (empty vector group) and non-transfected BMSCs (non-transfection group) were used as control group. RT-PCR, immunocytochemistry, and ELISA were performed to detect the expressions of TGF-β1 mRNA, TGF-β1 protein, and collagen type II. Results Successful construction of recombinant lentiviral vectors of porcine TGF-β1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-β1 recombinant lentiviral vectors. Green fluorescence was observed by laser confocal microscope. Western blot showed the optimal value of MOI was 70. The expression of TGF-β1 mRNA was significantly higher in TGF-β1 vector group than in empty vector group and non-transfection group (P  lt; 0.05). Immunocytochemistry results revealed positive expression of TGF-β1 protein and collagen type II in BMSCs of TGF-β1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-β1 protein still could be detected by ELISA in TGF-β1 vector group. Conclusion TGF-β1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-β1 protein can be observed, prompting BMSCs differentiation into chondrocytes.

Citation： LAI Jianming,LIN Jianhua,LIN Wenping,WU Chaoyang. CONSTRUCTION OF RECOMBINANT PORCINE TRANSFORMING GROWTH FACTOR β1 GENE LENTIVIRAL VECTOR AND ITS EXPRESSION IN BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(7): 849-844. doi: Copy