The Isolating Culture Of Neural Stem Cells From Rat's Fetus Brain And Transfection By Electroporation

Research background: Discovery and isolation of neural stem cells (NSCs) have changed the conventional idea that injured nervous tissue could not be regenerated. NSCs also have characters such as self-proliferation, multipotential differentiation and non-immunogenicity which make it an ideal cell carrier for transgenic therapy. Recent studies showed that therapy of NSCs transgenic transplantation for central nervous system diseases have obtained some efficacies in animal models of traumatic cerebral spinal cord injury, cerebrovascular disease, degenerative disease, hereditary metabolic disease and brain tumor. In 1989, vascular endothelial growth factor (VEGF), a carbohydrate protein, was first purified from astral cells of the cow pituitary gland follicle in vitro nutrient fluid by Ferrara. Its relative molecular weight is 34～45kD, and is comprised of two moleculars which are 17～22kD subunit the dimer and become the disulfide bond connection. VEGF promotes endothelial cell proliferation, accelerates the formation of new blood vessels, and increases the vasopermeability. Besides the effect on angiogenesis, VEGF may protect neural function and promote neurogenesis. The key factors of gene therapy are transfection efficiency and expression intensity of exogenous gene. There are many methods for mediating exogenous gene into eukaryocyte, such as viral gene delivery and non-viral gene delivery. Viral gene delivery has high transfection efficiency but low security and technology deficiency, which limits its application. Non-viral gene delivery includes cationic liposome and electroporation. Cationic liposome mediates the transfection of exogenous gene into NSCs, which efficiency is only 5%～15%.Objective: we isolated NSCs from the brain of SD fetal rats and sought for a high efficiency and non-viral gene delivery to transfect NSCs.Method: Firstly, we isolated NSCs from the brain of SD fetal rats, then NSCs were cultured and amplified by non-blood serum culture technique. NSCs and differentiated cells were identified using immunofluorescent histochemical methods. Secondly, recombinant plasmid pEGFP-N1/VEGF165 was constructed and certificated. Using green fluorescence protein(GFP) as the marker, pEGFP-N1 was transfected into NSCs using electroporation, and the tranfection rate was calculated by counting the NSCs with green fluorescent.Results: The cells isolated from brain tissue of fetal rats can proliferate for long time, both primary and passage culture of NSCs can express specific antigen of NSCs- Nestin, and after induced differentiation, the cells can express specific antigen of astrocytes-Glial fibrillary acidic protein (GFAP) and specific antigen of neurons-Neuron-specific enolase (NSE). The tranfection rates of electroporation were 20%～70%, and the average was 30%.Conclusion: Our experiment isolated NSCs which had the features of self-proliferation and differentiation potential from brain tissue of SD fetal rats. Recombinant plasmid pEGFP-N1/VEGF165 was constructed and certificated. Our experiment confirmed that electroporation was an efficient transfection method for NSCs, providing experimental basis for gene therapy in the future.