Effect Of Recombinant Human Erythropoietin On The Proliferation And Apoptosis Of Neural Stem Cells Derived From Adult Rat Spinal Cord In Vitro

Background&Objective:Spinal cord injury,which has a worldwide problem in post-injury neurological function recovery,is still lacking effective treatment methods in clinical practice.The use of exogenous cell transplantation to replace damaged nerve cells in the spinal cord is expected to become a cure for spinal cord injury.Neural stem cells(NSCs),derived from adult individual spinal cord central canal,as a source of transplanted cells for spinal cord injury have advantages such as low tumorigenicity,low immune rejection,and no ethical issues.However,when used to treat spinal cord injury by cell transplantation,problems such as insufficient number of transplanted cells and low cell survival rate in the transplanted area often happened.Therefore,how to promote the rate of proliferation and survival of neural stem cells cultured in vitro is crucial for the formation of a sufficient number of neural stem cells for transplantation in the treatment of spinal cord injury.The neuroprotective effect of erythropoietin(EPO)has received widespread attention in recent years.Some scholars also found that EPO has a certain role in promoting proliferation and anti-apoptosis of neural stem cells not derived from adult individual spinal cord in vitro.The use of EPO in promoting the growth and survival of neural stem cells derived from the spinal cord central canal of adult individuals in vitro would be of great benefit in the next step of transplantation therapy.But there have been few reports about the effect of EPO on the culture of neural stem cells derived from spinal cord central canal of adult individuals in vitro.The appropriate dose,appropriate time,and related mechanisms of EPO treatment are also inconclusive.This research,first,successfully isolated neural stem cells from the spinal canal of adult rats,and,then found that EPO of concentration of 5U/ml and 20U/ml,with a treatment time of 96h,plays a role in promoting proliferation,anti-apoptosis and regulating cell cycle.Finally,the mechanism of EPO on the physiological activities of neural stem cells derived from the spinal cord central canal of adult rats was also explored.The main methods used in this stduy are as follows:1.Neural stem cells were extracted from the spinal cord central canal of adult rats according to previous methods and identified by cell immunofluorescence staining.2.The CCK8 assay was used to preliminarily screening the suitable concentrations and treatment time of EPO on neural stem cells derived from the central canal of adult rats spinal cord.Neurosphere calculation and cell count were also used to confirm the appropriate concentration and treatment time.3.EdU detection,TUNEL assay and flow cytometry were used to verify that appropriate concentrations of EPO would affect the proliferation,apoptosis,and cell cycle of neural stem cells derived from the central canal of adult rats spinal cord at appropriate treatment time.Finally,real-time quantitative PCR was carried out to determine the changes in the expression of related genes.4.The data was processed by using SPSS20.0 software(IBM Corp.);and the results were measured as mean±standard deviation(X±SD)or mean X.One-way analysis of variance(One-Way ANOVA)was used for comparison between groups.A P-value of less than 0.05 was considered statistically significant.Result:In this experiment,neural stem cells were successfully extracted from the central canal of adult rats spinal cord.The results of CCK8 detection showed that concentrations of EPO of 5U/ml and 20U/ml may be an appropriate concentration to affect the proliferation of neural stem cells derived from the central canal of adult rats spinal cord,and the appropriate processing time may be 96h.Subsequently,the results of neurosphere calculations,cell counts,EdU assays,and flow cytometry tests further validated the results of CCK8 detection;TUNEL assays also confirmed that using EPO at a suitable concentration and time can downregulate the apoptosis of neural stem cells.Finally,the results of real-time quantitative PCR also proved that EPO with appropriate concentration and treatment time can upregulate genes related to proliferation,anti-apoptosis and cell cycle regulation.Conclusion:The concentration of EPO of 5U/ml and 20U/ml may be the appropriate concentration to promote the effects of proliferation,anti-apoptosis and cell cycle regulation of neural stem cells derived from the central canal of adult rats spinal cord;and the appropriate treatment time may be 96h.