[Experimental study on transforming growth factor beta3 gene transfecting into marrow mesenchymal stem cells in rabbits]

Lin Qiu, Xianqing Jin, Xiaoji Luo
Chinese Journal of Reparative and Reconstructive Surgery 2006, 20 (9): 940-3

OBJECTIVE: To construct recombinant adenovirus vector containing human transforming growth factor beta 3 (TGF-beta3), which was transfected into marrow mesenchymal stem cells (MSCs) and to observe its expression.

METHODS: The cDNA TGF-beta3 was integrated into the shuttle vector of pAdTrack-CMV and recombinated with adenovirus skeleton vector pAdEasy-1 by homologous recombination. Then the product was transfected into package cell HEK293 by lipofectamine and the recombinant adenovirus expressing the TGF-beta3 gene was generated. The rabbit's MSCs were isolated, cultivated, purified, and then transfected with recombinant adenovirus containing the TGF-beta3 gene. The green fluorescence protein expression was observed after 10 days, and the TGF-beta3 expression was observed in MSCs transfected by recombinated adenovirus with TGF-beta3 gene after 4 days.

RESULTS: PCR showed that TGF-beta3 cDNA was inserted into the recombinant adenoviral plasmid. The recombinant virus vectors with TGF-beta3 gene were collected by the packaging HEK293 cells. The fusion rate of MSCs was 70%-80% with an intensive adhesion and uniform shape after the cultured 10th day. Fluorescent microscopy and immunocytochemistry demonstrated that TGF-beta3 was expressed in MSCs.

CONCLUSION: Successful construction of human TGF-beta3 recombinant adenovirus and its expression in MSCs provide a basis of research for the gene therapy of wound healing.

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