Experimental Study Of The Rat Mesenchymal Stem Cells Transfected With Sonic Hedgehog Gene

Background:Sonic Hedeghog (SHH) gene by upregulating the expression of a variety of angiogenic factors to participate in angiogenesis. Endogenous SHH pathway will also be activated after myocardial ischemia. Based on the role of SHH gene in the cardiovascular system development and the promotion of tangiogenesis, SHH gene may therefore as a potential treatment strategy used in myocardial ischemia. Mesenchymal stem cells (MSC) can muscle cell and endothelial cell differentiation, the promotion of angiogenesis and myocardial remodeling in myocardial ischemic injury, improve heart function. In addition, MSC can also accept the variety of gene transfer vectors and maintain good genetic stability even after in vivo differentiation. So it can also serve as target cells for gene therapy.Objective:Evaluate the feasibility of genetic modification of MSC with SHH transgene.Methods:Wistar rat SHH gene cloning and sequencing. Construction PCDNA3.1-SHH eukaryotic expression vector, MSC of Wistar rat was isolated by density gradient centrifugation and purified on the basis of their ability to adhere to plastic. Detections of cell surface antigens, including CD34, CD45, CD44 and SH3, were performed using flow cytometry. Transfection SHH into MSC by Nucleofector and identify the expression of SHH by Western-blot analysis.Results:RT-PCR amplify the rat SHH gene coding sequence (CDS) fragment. Cloning by restriction digestion and sequencing of rat SHH gene sequence consistent with the Genebank. MSC were CD34, CD45, CD44+ and SH3+. Western-Blot test confirmed Nucleofector ways to transfection SHH gene into the rat MSC, and was expressed. But MSC transfected with empty plasmid expression was not detected.Conclusions:Recombinant Eukaryotic expression vector PCDNA3.1-SHH can expression in rat MSC. The experiment demonstrated that SHH had successfully expressed in MSC. To further study the SHH gene transfer MSC transplantation on rat model of myocardial ischemia and cardiac function to provide an experimental basis.