[Adenoviral vector-mediated human hepatocyte growth factor gene transfection for its high expression in rabbit bone marrow-derived mesenchymal stem cells]

Qian Wen, Li Ma, Dan Jin, Jian-de Cui, Wei Luo, Xiao-ning Wang
Nan Fang Yi Ke da Xue Xue Bao, Journal of Southern Medical University 2007, 27 (11): 1627-30

OBJECTIVE: To obtain high expression of human hepatocyte growth factor (HGF) in passaged rabbit bone marrow-derived mesenchymal stem cells (BMSCs) via recombinant adenovirus vector mediated HGF gene transfection, and explore the feasibility of this strategy for local treatment of avascular necrosis of the femoral head (ANFH).

METHODS: HGF gene was subcloned into the adenovirus shuttle plasmid pDC316, and the products were co-transfected into HEK293 cells with the helper plasmid pBHGlox deltaE1,3Cre. The recombinant adenovirus Ad-HGF was generated by homologous recombination of the 2 plasmids in HEK293 cells. After PCR identification, Ad-HGF was amplified and purified and its titer measured by TCID50 assay before transfected into the second passage of rabbit BMSCs. The transcription and expression of HGF gene in the transfected BMSCs was detected by RT-PCR, in situ hybridization, and immunological histochemistry.

RESULTS: Ad-HGF was successfully constructed with a titer of 2.6x10(10) TCID50/ml. The expressions of HGF mRNA and protein were confirmed in the transfected BMSCs.

CONCLUSION: Ad-HGF transfection allows efficient expression of HGF protein in rabbit BMSCs, and this success may facilitate further study of local treatment of ANFH using HGF-expressing BMSCs.

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