[Construction of Lentivirus Vector Carrying HGF and Exploration of Transfection Condition of Rat Adipocyte-derived Mesenchymal Stem Cells].

OBJECTIVE

This study was to construct the lentivirus vector carrying hepatocyte growth factor (HGF) gene and to explore the condition for transfecting the adipocyte-derived mesenchymal stem cells (ADSC) by HGF lentivirus.

METHODS

The target gene was obtained from plasmid carrying HGF gene by PCR and was cloned into GV287 vector. The recombinant GV287-HGF vector plasmid and lentivirus-packing plasmid were co-transfected into 293 T cells to generate HGF lentivirus, and the virus titer was assayed, then the ADSC were transfected by using recombinant HGF lentivirus, and the optimal multplicity of infection (MOI) was detected.

RESULTS

The PCR product of HGF gene was consistent with expectant sizes, suggesting that the electrophoretic result of recombinant GV287-HGF plasmid PCR product was correct. The sequencing analysis of cleaved product showed consistance of obtained results with the sequences of target gene, suggesting correct construction of recombinant lentivirus carrying HGF gene. The ELISA showed that the virus tilter was 5×10(8) TU/ml. The optimal MOI for transfecting ADSC with recombinant lentivirus carrying HGF gene was 50.

CONCLUSION

The lentivirus vector expressing human HGF gene has been constructed, and transfected the ADSC succesfully. This study lays a foundation for further stadying the ADSC over-expressioning HGF, treating the radiation damage of bone marrow and impartant internal organs.