The Effect Of Analog Neural Regeneration Microenvironment By VEGF-165and Matrigel Basement Membrane Matrix On Hippocampal Neural Stem Cells

ObjectiveNeural stem cell transplantation provides a new therapeutic approach for central nervous system damage. But in recent years, It’s found that the survival rate of neural stem cells is low and only a little of NSCs can differentiate into neurons after exogenous neural stem cell transplantation. All these problems may be solved in a new direction after the concept of blood vessels-neurogenesis and neurogenesis microenvironment was proposed. This time, we will extract neural stem cells from neonatal rat in vitro culture based investigate and research neural stem cell growth and differentiation in the analog neurogenesis microenvironment by VEGF-165and Matrigel basement membrane matrix.MethodThis research is base on the neural stem cells extracted from the hippocampus of the newborn SD rats (within24hours),and propagate NSCs through vitro culture and identifie it by Nestin immunohistochemical staining. Seed the NSCs that cultured successfully on24-well plate coated by poly-lysine and Matrigel basement membrane matrix, and give different culture medium (normal neural stem cell culture medium, fetal bovine serum culture medium, VEGF-165induced differentiation culture medium and VEGF&fetal bovine serum culture medium) to induce differentiation. For a week’s induction of differentiation,ditect nestin, β-tubulin III, GFAP in the cells after induction by immunofluorescence staining, and observate the condition of the proportion of positive cells about three proteins, and compared the differentiation of neural stem cells before and after the application of Matrigel basement membrane matrix, to study the effection of VEGF-165and Matrigel basement membrane matrix to neural stem cells.Results:1. The culture of neural stem cell:It has cultured in vitro about one week after cells were extracted from the neonatal rat hippocampus to form balls of cells of hundreds cells. Using trypsin and mechanical separation method to blown off the balls of cells to passage, cells grow well and can proliferate stably. After immunohistochemical staining of nestin, the balls of cells overwhelming stained strongly positive reaction. So, the cells extracted from neonatal rat hippocampus are neural stem cells.2. The differentiation after VEGF-165-induced:Through different medium induce the growth and differentiation of neural stem cells, the effection of VEGF-165can be infered roughly for the growth and differentiation of neural stem cells, that VEGF-165can regulate the proliferation and differentiation of neural stem cells, and possibly can promote NSCs to divide into neurons. Vascular endothelial growth factor (VEGF) may be a contributing factor to the proliferation and differentiation of neural stem cells, has an important significance for the study of the generation and differentiation of neural stem cells.3. The combined effects of Matrigel basement membrane matrix and VEGF-165: Matrigel basement membrane matrix can provide an ideal environment of three dimensional for the proliferation and differentiation of neural stem cells, and can promote the differentiation of neural stem cells. After combinating with VEGF-165,the proportion of NSCs divided into neurons is improved, and the structure between cells is more similar with the structure of the organization in vivo. This seems important for neurogenesis.ConclusionIn summary, the combination of Matrigel basement membrane matrix and VEGF-165can simulate the microenvironment of neurogenesis, and can provide a appropriate environment for growth and differentiation of neural stem cells. In some degree, the combined application can improve the differentiation trend of neural stem cells divided into neuron, and form a three-dimensional cell connection which is more approximate normal tissue structure than general adherent culture. Through this study, Matrigel basement membrane matrix and cytokines such as VEGF-165can well imitate neurogenesis microenvironment,and can provide some new ideas for the low survival rate of NSCs and low neuronal differentation in transplantation.