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Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots
Published on: May 21, 2013
[Experimental study on transforming growth factor beta3 gene transfecting into marrow mesenchymal stem cells in
Lin Qiu1, Xianqing Jin, Xiaoji Luo
1Department of Burns and Plastic, Children's Hospital, Chongqing University of Medical Sciences, PR China. qiulin118@gmail.com
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.
