[Construction of recombinant porcine transforming growth factor beta1 gene lentiviral vector and its expression in bone marrow mesenchymal stem cells]
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Published on: October 30, 2018
Jianming Lai1, Jianhua Lin, Wenping Lin
1Department of Orthopedics, Longyan First Hospital Affiliated to Fujian Medical University, Longyan Fujian, 364000, PR China.
Objective:
To construct recombinant lentiviral expression vectors of porcine transforming growth factor beta1 (TGF-beta1) gene and transfect bone marrow mesenchymal stem cells (BMSCs) so as to provide TGF-beta1 gene-modified BMSCs for bone and cartilage tissue engineering.
Methods:
The TGF-beta1 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-beta1 recombinant lentiviral vectors (TGF-beta1 vector group) respectively at multiplicity 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-beta1 mRNA, TGF-beta1 protein, and collagen type II.
Results:
Successful construction of recombinant lentiviral vectors of porcine TGF-beta1 gene was identified by PCR and gene sequencing, and BMSCs were successfully transfected by TGF-beta1 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-beta1 mRNA was significantly higher in TGF-beta1 vector group than in empty vector group and non-transfection group (P < 0.05). Immunocytochemistry results revealed positive expression of TGF-beta1 protein and collagen type II in BMSCs of TGF-beta1 vector group, but negative expression in empty vector group and non-transfection group. At 21 days after transfection, high expression of TGF-beta1 protein still could be detected by ELISA in TGF-beta1 vector group.
Conclusion:
TGF-beta1 gene can be successfully transfected into BMSCs via lentiviral vectors, and long-term stable expression of TGF-beta1 protein can be observed, prompting BMSCs differentiation into chondrocytes.
