Infection Of Endothelial Progrnitor Cellls From Rat With Adeno-Associated Virus Containing β-NGF Gene And The Effect On The Proliferation Of EPCs

Backgound:With the development of industrialization, there is an upward trend in spinal cord injury incidence rate in our country, tens of thousands of people suffer from spinal cord injury each year. But there is lack of effective treatments for spinal cord injury since promoting neural restoration has been a difficult problem. Nowadays, gene therapy has become a new trend for disease therapy and brought promise for some refractory diseases. It is the key point to choose the proper cell, gene and vector,the experiment takes a good use of endothelial progenitor cells(EPCs) which can promote new blood formation, nerve growth factor(NGF) which can promote restoration of the damaged nerve and adeno-associated virus vector which is confirmed safe and reliable.It is confirmed by this experiment that the objective gene β-NGF can express with a high-efficiency in the transfected endothelial progenitor cells and promote the proliferation of primary cultured EPCs.Objective:To investigate recombinant adeno-associated virus (rAAV) mediated nerve growth factor (β-NGF) transfecting rat bone marrow derived endothelial progenitor cells(EPCs) in vitro, and the effect on EPCs’ proliferation. And to exam the effectiveness and feasibility of β-NGF gene transfected into endothelial progenitor cells (EPCs) from rat bone marrow for gene therapy.Methods:The EPCs were isolated, cultured and identified from the bone marrow of rats. Empty vector (rAAV-GFP) or β-NGF gene (rAAV-GFP-β-NGF) was transferred into EPCs by the recombinant adenovirus-associated virus (rAAV). We examined the transfection efficiency by GFP fluorescence, demonstrated the secreted β-NGF Protein concentration in the supernatant by ELISA, and studied its effect on the proliferation of primary cultured EPCs with the methods of MTT.Results:Rat EPCs were isolated and cultured successfully in vitro and were identified positive by the function of cells and immunofluorescence staining. The EPCs can be infected directly by the recombinant adenovirus-associated virus containing β-NGF gene with an efficiency of 65.30%.β-NGF Protein were detected in culture medium after EPCs were transfected by rAAV-GFP- β-NGF, which reached to a high level at ten days after gene transfection. Furthermore, After transfected β-NGF gene, EPCs could promote the proliferation. The proliferation of β-NGF groups were significantly greater than that of control groups (P<0.05).Results:After successful transfection on EPCs, the objective gene β-NGF can effective express in the transfected cells and promote the proliferation of primary cultured EPCs, which provides the support for further cure of the spinal cord injury disease with gene-stem cell therapy.