The Effects Of Fasudil Hydrochloride On Differentiation Of Neural Stem Cells In Vitro

BackgroundNeurocyte is the terminal cell which lack the regeneration capacity in traditional view, the generation of neurocyte only restrict at the transient period of embryo and postnatal, when the central nervous system (CNS) disease as ischemia,trauma and degeneration damage neurons, the neurons can not regeneration to creation the new synapses and postinjury its function is difficult to recover. But study indicate that, however in embryo stage or adulthood, the central nerve system of mammals have neural stem cells (NSCs) which have the capability of self-renewal and multi-directional differentiation, and this is the hope of the treatment of nervous system injuries or degeneration. But, now there has not the method which can control the generation,differentiation,immigration of NSCs effectively,make them to reach the concerted area and differentiate to the needed cell. And the inhibiting factor which present around the injuried spot can prevent the NSCs into never cells,but promote into gliocyte.So, the mechanism of the generation,differentiation and immigration of NSCs is a hotspot of the study nowadays. Protein RhoA family is an important regulatory molecules of cytoskeleton actin, and as a signaling molecules participate in many cell signal transductions which are connection with cytoskeleton. Rho-kinase conduction path has an important accommodation on cell's divide and conglutination,differentiation and migration,cytoskeleton' regulation,muscular cell contraction,tumor cell invade etc. Lately study show that Rho-kinase is closely related to many nervous system diseases,and inhibit Rho-kinase activity can prevent effectively and treat nervous system diseases,so Rho-kinase inhibitor become a target point of new nervous system drugs' research. Fasudil Hydrochloride(six hydrogen-1-(5-isoquinolylsulfonyl)-1H-1,4-dinitrogencycloheptane, Fasudil, named HA1007 also) is a frequently-used Rho-kinase inhibitor in clinic, and generally used in cerebrovascular disease. This study is to observe the effects of Fasudil Hydrochloride on differentiation of neural stem cells in vitro, and it can induce the NSCs' directional differentiation or not, and this have the important significance to treat the nervous system injuries and degeneration diseases by the therapy of the transplantation of NSCs.Purpose1.Isolate and cultivate the pure neural stem cell.2.Study the effects of Fasudil Hydrochloride on differentiation of neural stem cells in vitro.Method1. Neural stem cells were extracted form brain of newborn Wistar rats within 24h and cultured using the method of suspension culture. Then subculture and the stem cell differentiation experiments were done. Immunofluorescence were used to identify the neural stem cells and the cell surface antigen after adherence through Nestin, neuron specific enolase(NSE) and Glial fibrillary acidic protein(GFAP) staining.2.The third generation stem cells were used for the experiment. Final concentration of 50μmol/L, 100μmol/L and 200μmol/L Fasudil hydrochloride was added respectively to for the experimental group. Not adding to Fasudil hydrochloride was for the control group. After 30 min of intervention, the cells were continued to culture using the neural stem cell differentiation culture medium. Then the morphology of neural stem cells were observed under the inverted microscopy.3.Observe the expression of the cell's NSE,GFAP after differentiation by immunohistochemisty, and calculate the percentage of the positived cell in every group by double blind method, test the percentage of the NSE-positived cell by flow cytometer.4.Test the survival ratio of the cells after differentiation by the method of AO-EB staining and assaying the activity of LDH.Result1.Neural stem cells extracted from the newborn rats have the potential of self-renewal and multi-directional differentiation. The immunohistochemistry staining of Nestin was positive and the differentiated cell can express NSE(neurons'marker) and GAFP(neuroglia cells'marker).2. We found that the experimental group can differentiate more neuron-like cells and more immunohistochemisty stained NSE-positived cells were observed comparing with the control group. There were significant differences(P<0.05).3.Comparing with control group, the experimental group can differentiate more neuron-like cells in flow cytometry detecting, and There were significant differences(P<0.05). Athough we found that the higher concentration of Fasudil hydrochloride, the more neurons can be differentiated by the neuronsphere, but there were no significant differenes between the groups(P>0.05).4.AO-EB staining: observed under the inverted microscope, the morphology of the majority of cells in the Fasudil hydrochloride group was plump, with green cytoplasm, higher green nuclear, regular nuclear morphology of round or ellipse, and few orange nuclears. But in the control group, the red nuclear number increased obviously comparing with the experimental group. LDH activity assay showed that the neural stem cells begin to differentiate in the second day, LDH activity assay in the control group was significantly higher than the experimental group and the difference was statistically signigicant(P<0.05).Conclusion1.Neural stem cells were successfully isolated from the newborn rats, and were proliferated, subcultured and induced to differentiate to neurons(positive NSE) and glial cells(positive GFAP) in vitro.2.Fasudil Hydrochloride can promote neural stem cells differentiation into neurons.3.Fasudil Hydrochloride play protective effect to never cell,and the apoptosis rate after differentiated is lower in the experimental group compared to the normal control group.