| A variety of reasons cause the decrease of blood perfusion in tissues and organs for a certain degree and for lasting a certain period of time,leading to the ischemic injury of tissue cells.The brain is the most sensitive organ to ischemia and hypoxia,and it mainly relies on glucose aerobic oxidation to provide energy,and cerebral ischemia can lead to severe irreversible injury.Ischemic cerebrovascular disease is one of the most common diseases that harm human health,and it has the characteristics of high morbidity,high mortality and high disability rate.In the traditional view,the death of the nerve cells in the ischemic area is irreversible,and mature nerve cells around the infarct area can not be regenerated to compensate for the dead nerve cells.Cerebral ischemia has Poor prognosis.The research shows that nerve cells which are in the acute phase of ischemic brain injury are mainly necrotic occurring in the central area of the infarct,and that delayed death of nerve cells is mainly apoptosis occurring in the Ischemic penumbra.So the key to treatment is to save the dying nerve cells in the ischemic penumbra.Anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax plays an important role in the process of nerve cells apoptosis.Anti-apoptotic protein Bcl-2 and Pro-apoptotic protein Bax play an important role in the process of nerve cells apoptosis.We can inhibit the apoptosis of nerve cells by up-regulation of Bcl-2 and Down-regulation of Bax.Neural stem cells are the progenitor cells of the nervous system,which can be differentiated into nerve cells and glial cells.This physiological characteristics provides a theoretical basis for nerve regeneration and repair.In recent years,with the maturation of NSCs in vitro culture,exogenous NSCs transplantation has been used in the treatment of a variety of nervous system injuries and degenerative diseases.Buyang Huanwu Decoction is a classic prescription for the treatment of ischemic stroke.It plays an important role in improving the sequelae of apoplexy and reducing the rate of disability.A large number of studies have shown that both neural stem cell transplantation and Buyang Huanwu Decoction can promote the repair of nerve injury and reduce the apoptosis rate of nerve cells.However,it is still unknown whether giving Buyang Huanwu Decoction after the transplantation of neural stem cells in cerebral ischemia reperfusion rats can further promote the recovery of neurological function and further reduce neuronal apoptosis.Therefore,this experiment combined Buyang Huanwu Decoction and NSCs transplantation to explore the neuroprotective effect in cerebral ischemia reperfusion rats.The First Part Comparative study on the extraction and culture method of neural stem cellsObjective:To explore the best method of neural stem cell extraction,culture and identification,and provide a technical reference for the basic research of neural stem cells.Methods:Comparison of different extraction and culture methods of neural stem cells:Observation the rate of ball of neural stem cells and the stability of neural stem cells in undifferentiated state by extraction of fetal and neonatal rats cortex,using different types of medium,different inoculation density,Different culture methods,Different methods of changing liquid and different passage methods.Detection of neural stem cells activity by Varioskan LUX Multimode Microplate Reader.Results:1.Fetal rats of 14d brain cortex had higher proportion of neural stem cells,less other cells and more neurospheres than newborn rats of 24h.2.Neural stem cells could be stabilized in undifferentiated state by using serum-free medium,while most of the neural stem cells differentiated into neurons and glial cells by using serum medium.3.Neural stem cells,seeding at 1.0×109·L-1,had a large number of neurosperes and were in good condition.4.Suspension culture was beneficial to form a stable neurosphere and keep the neural stem cells in an undifferentiated state than adherent culture.5.The state of neurosphere by changing half of the medium and adding medium without discarding it better than replacing all medium.6.The neurospheres could be separated into single cells by mechanical blow in primary,generation and second generation of neurospheres cultured in serum-free medium.Whlie the percentage of viable cells in neural stem cells was the highest digested with stem cell lysates after three generations and the neurospheres re-formed were better.7.Neural stem cells activity of 14d fetal rat Accutase digestion group was significantly higher than that of the other three groups,the difference is significant?P<0.05?.Summary:Neural stem cells proliferate and divide well,has higher rate of ball formation,condition of neurosphere well and can maintain a stable undifferentiated state by extracting the cerebral cortex of 14d fetal rats,using serum-free medium,inoculating into a 25 cm2 flask at a density of1.0×109·L-1,taking the suspension culture?Adding the medium 1-1.5ml every 2-3day and passage every 6-7 day?.The Second Part The protective effect of nerve function of Buyang Huanwu Decoction on cerebral ischemia reperfusion rats after neural stem cell transplantationObjective:To explore the protective effect of nerve function of Buyang Huanwu Decoction on cerebral ischemia reperfusion rats after neural stem cells transplantation.Methods:Extracting the cerebral cortex of 14d fetal rats,isolation and culture and identification of neural stem cells.60 male SD rats were divided into 5groups randomly?n=12?:Sham operation group,Model group,BYHWD group,Transplant group,BYHWD+Transplant group.In addition to the sham operation group,we used modified thread bolt method to establish middle cerebral artery occlusion model for other groups.BYHWD group and BYHWD+Transplant group were given Buyang Huanwu Decoction(14.3g·kg-1·d-1)by gavage after the rats being sober.NSCs were transplanted to Cerebral ischemia side striatum and cerebral cortex in rats brain after making the model 24 hours later.Zea Longa neurobehavioral behavioral score was used to observe neural function defect,and TTC staining to detect the volume of cerebral infarction,and Nissl staining to detect Nissl body integrated optical density?IOD?,and Immunohistochemical staining to detect expression of Bcl-2 and Bax.Results:The sham operation group had no nerve function defect,and cerebral infarction was not found in TTC staining,and Nissl body integrated optical density was higher,and the number of Bcl-2 positive cells and Bax positive cells was less.Compared with the sham operation group,the nerve function defect was significantly aggravated,and the volume of cerebral infarction increased significantly,and Nissl body integrated optical density is obviously reduced,and the ratio of Bcl-2 to Bax is significantly reduced in the model group?P<0.05?.Compared with the model group,the nerve function defect was reduced,and the volume of cerebral infarction was reduced,and Nissl body integrated optical density increased,and the ratio of Bcl-2 to Bax increased in BYHWD group,Transplant group and BYHWD+Transplant group?P<0.05?.Compared Transplant group,the nerve function defect was reduced,and the volume of cerebral infarction was reduced,and Nissl body integrated optical density increased,and the ratio of Bcl-2 to Bax increased in BYHWD+Transplant group?P<0.05?.Summary:Buyang Huanwu Decoction can reduce the degree of nerve function defect after NSCs transplantation in cerebral ischemia reperfusion injury rats,reduce the volume of cerebral infarction,increase Nissl body integrated optical density,increase the ratio of Bcl-2 to Bax,and play the role of neuroprotection.Conclusion:1.Neural stem cells proliferate and divide well,has higher rate of ball formation,condition of neurosphere well and can maintain a stable undifferentiated state by extracting the cerebral cortex of 14d fetal rats,using serum-free medium,inoculating into a 25 cm2 flask at a density of1.0×109·L-1,taking the suspension culture?Adding the medium 1-1.5ml every 2-3day and passage every 6-7 day?.2.Buyang Huanwu Decoction can reduce the degree of nerve function defect after NSCs transplantation in cerebral ischemia reperfusion injury rats,reduce the volume of cerebral infarction,increase Nissl body integrated optical density,increase the ratio of Bcl-2 to Bax,and play the role of neuroprotection. |
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