Effect Of Endothelial Progenitor Cells On Proliferation, Differentiation Of Neural Stem Cells And Expression Of Notch Signal Molecules

Objective:Ischemic cerebrovascular disease has become one of the main reasons for causing death and Mutilation in humanbeing. It consists of two main pathological lesions: the loss of neuron and regional blood circulation. The repairs of blood supply and neural function are the two main aspects in its clinical therapy.Conventional idea considers that agagenesis is never occurred in central nervous system in adult mannal , it is difficult to repair if the loss of neuron occurred. But in 1990s of last century, the discoveries of neural neogenesis and neural stem cells changed the conventional idea that nerves can not be repaired after brain damage. In recent years ,neural stem cells can be activated by some dissoluble cell damage materials occurred in the process of various kinds of brain damages such as cerebral ischemia, epilepsy,and brain wounds, and can be a selective repair to cortical neuron and bindweb losed through neural neogenesis. The regulation mechanism of generation and differentiation of neural stem cells is growing to search hot spot of neuroscience.The mechanism of generation and differentiation of neural stem cells is complicated and various, Among which notch signal pathway is a conventional way to regulate cell proliferation and differentiation. Notch signal pathway determines whether neural stem cells choose to generate or differentiate to neuron, and is correlted with the proportion of neuron and colloid differentiated from neural stem cells.With the theory of endothelial progenitor cells established , it is paid close attention to that endothelial progenitor cells are applied to treat blood vessel's neogenesis and promote the rebuilding of blood vessels in the area of cerebral ischemia. Some research found that endothelial progenitor cells can be mobilized and released from bone marrow to peripheral blood whild ischemic cerebrovascular disease occurred; the cells migrate to the area of cerebral ischemia and participate rebuilding of blood vessels and the revascularization of ischemic tissue, make the capillary density up-regulated and infarct size contracted and neural function improved.Neurogenesis is correlated closely with vasculogenesis . The recovery of blood supply is the base of the recovery of nerves. Endothelial progenitor cells is not only correlated closely with the recovery of blood supply, but also promote endogenous neural stem cells immigrate to ischemic brain tissue and participate repairing. It is hinted that the transplant of endothelial progenitor cells or the transplant of both endothelial progenitor cells and neural stem cells meet with positive perspective in treating ischemic cerebrovascular disease. However, the effect of endothelial progenitor cells on neural stem cells is not clear, so we applied co-culture model in vitro to research the effect of endothelial progenitor cells derived from bone marrow on the proliferation and differentiation of neural stem cells, especially on Notch signal pathway, so as to approach the mechanism how endothelial progenitor cells regulate the proliferation and differentiation of neural stem cells and to establish the rationale that the transplant of endothelial progenitor cells or the transplant of both endothelial progenitor cells and neural stem cells treat central nervous system disease.Methods:1.Rat endothelial progenitor cells were isolated and cultured by adherence screening method , identified by morphology, immunofluorescence with CD34,CD133, FLK-1,the factor ofⅧ,and RT-PCR of the genes:CD34,CD133,FLK-1,the factor ofⅧ,eNos.2.Mouse neural stem cells were cultured by Neural stem cell suspension method , and identified by immunofluorescence with nestin.3.We build Transwell co-culture system , and Experimental cells were randomly divided into five groups:①The group of endothelial progenitor cells coculcured with neural stem cells (EPCs+NSCs):EPCs applied to experiment were first passage cells. while NSCs were third passage cells. While colculcured , EPCs were were seeded in removable Transwell inserts(upper or apical chamber ), NSCs in the lower chamber.②The group of endothelial progenitor cells colculcured with neural stem cells treated by SU5416 [EPCs+(SU5416+NSCs)]:After treated by SU5416(10μM)for 6 hours, neural stem cells in the lower chamber were colculcured with endothelial progenitor cells in removable Transwell inserts.③The group of endothelial progenitor cell colculcured with neural stem cells treated by DAPT [EPCs+ (DAPT+NSCs)]: EPCs were were seeded in removable Transwell inserts, NSCs in the lower chamber after treated by DAPT(10μM).④The groups of neural stem cells treated by DAPT(DAPT+NSCs):the equality medium without EPCs was added into the upper chamber, and NSCs in the lower chamber after treated by DAPT(10μM).⑤The control group(NSCs):: the equality medium without EPCs was added into the upper chamber,NSCs in the lower chamber.4. mRNA of five groups of NSCs extracted at three different times:co-cultured after 2h,6h,12h.Then,RT-PCR were employed to assay the expressions of Notch pathway signal molecules: notch1,mash1 and HES1,and of neuronal differentiation of neural stem cells.Results:1.Endothelial progenitor cells manifest the best representation,the cobblestone and alignment structure of attached cells were observed The cells were identified by cell shape, positively staining for CD34,CD133,FLK-1,the factor ofⅧ,and RT-PCR of the genes:CD34,CD133,FLK-1,the factor ofⅧ,eNos.2.NSCs were isolated from neocortical tissue of SD rat, were identified by cell shape , immunocytochemistry.3.The diversities of the related genes with the proliferation and differentiation of neural stem cells and with notch signal pathway(1)Endothelial progenitor cells can enhance the expression of ki67 gene and nestin gene. Compared with the group of [EPCs+(SU5416+NSCs)], the group of (EPCs+NSCs)can make the expression of ki67 gene and nestin gene more up-regulated. It demonstrated that endothelial progenitor cells can promote the expression of ki67 gene and nestin gene ,which is related with endothelial growth factor secreted from endothelial progenitor cells.(2)Endothelial progenitor cells made the expression ofβ-Tubulin gene up-regulated, and the expression of GFAP gene down-regulated . the expression ofβ-Tubulin gene were up-regulated with the diversity levels , and the expression of GFAP gene down-regulated in the groups: (EPCs+NSCs),[EPCs+(SU5416+NSCs)] and [EPCs+(DAPT+NSCs)] ,especially in the group of(EPCs+NSCs). It indicated that endothelial progenitor cells can promote neuronal differention of neural stem cells, which is related with the regulation of notch signal pathway and endothelial growth factor.(3)In the groups of (EPCs+NSCs)and [EPCs+(SU5416+NSCs)], the expression of hes1 gene of neural stem cell were down-regulated while the expression of mash1 gene up-regulated.In a word, endothelial progenitor cells enchance the expression of ki67 gene and nestin gene which related the proliferation of NSCs ,and the expression ofβ-Tubulin gene related differentiation of NSCs,while down-regulate the expression of GFAP gene related glial differentiation of NSCs.the effect of EPCs is closely related with the regulation of the genes of notch signal pathway and endothelial growth factor.Conclusion:Endothelial progenitor cells can promote the generation and neuronal differention of neural stem cells,which related with the regulation of the genes of notch signal pathway and endothelial growth factor.