Neural Stem Cell Transplantation Is Associated With Inhibition Of Apoptosis,Bcl-xL Upregulation,and Recovery Of Neurological Function In A Rat Model Of Traumatic Brain Injury

Objective:In recent years,the morbidity and disability rate of Traumatic brain injury(TBI)have increased year by year.However,TBI pathogenesis is a complex process,so there is guidelines for treatment to reduce the disability and mortality rate of TBI worldwide.Explore the possible mechanism of neural stem cell(NSCs)transplantation in the repair of TBI and whether Bcl-xL can be used as a target of treatment.Method:1.TBI model:20 SD rats were divided into false surgery group and TBI group,each group was 10,and mNSS score was performed 7 days after surgery.We used damaged brain tissue of 5 rats of each group for general appearance observation and TUNEL test;The other five rats were raised to 14 days for mNSS score.2.We used 5 embryonic mice(embryonic day 12 to 14)with transgenic green fluorescent protein for NSC isolation.3.Human HSV-1 deficiency virus was used as vector skeleton,human cytomegalovirus as promoter WPRE and PolyA as accessory,and Vero cells were used to transfect the recombinant Bcl-xL to construct human HSV-1 transporter.4.We used 60 SD rats for experiment.First,30 SD rats were randomly divided into three experimental groups:TBI +culture medium injection group(TBI),sham operation group(sham)and TBI+ NSC transplantation group(NSCs),with 10 rats in each group.Second,another 30 rats were randomly divided into 3groups:TBI group(TBI plus HSV-1-negative vector,i.e.,TBI+HSV-1-NC vector),NSC group(TBI plus NSCs,i.e.,TBI + NSCs),and Bcl-xL-ORF group(TBI plus Bcl-xL overexpression,i.e.,TBI+ HSV-1-exBcl-xL),with 10 rats in each group.After 7 days of operation,we evaluated mNSS score and molecular biology.The other half rats were fed until 14 days after operation,we evaluated mNSS score only.5.The neuronal apoptolo s in the host brain were detected by TUNEL staining.The expression of Bcl-xL,FasL,Bcl-2Bax,Caspase3 were detected by RT-PCR and Western-blot.Identification and Differentiation of NSCs In Vitro were detected by immunofluorescent staining.Result:1.Vesus sham group,TBI group had an increased mNSS score for 7 days and 14 days(P<0.05),which was mainly manifested as spasmodic paralysis and balance dysfunction.Seven days after the operation of the TBI group,A large number of nerve cell apoptosis was seen in the damaged brain tissue,and only a small amount of nerve cell apoptosis was seen in the sham surgery group(P<0.05).2.After 12 hours of NSCs culture,the cells were small round or elliptical clear single cells;after 3 days,the single cells gathered to form neurospheres;after three generations of culture,the Nestin labeled positive cells were undifferentiated NSCs;,NeuN labeled positive cells were neuronal cells and GFAP-labeled positive cells were astrocytes.3.The mNSS scores in the TBI construction group were significantly higher than those before the operation on the 7th and 14th day after operation(P<0.05),mainly for spastic paralysis of limbs and disturbance of balance function(P<0.05).The mNSS score decreased 14 days after operation and 7 days after operation.Brain edema and hemorrhage were visible to the naked eye.7 days after operation,a large number of neuronal apoptosis was found in the TBI construction group,but only a small number of neuronal apoptosis was observed in the sham operation group(P<0.05).4.The mNSS scores of 7 and 14 days after NSCs transplantation were lower than those in sham operation group and TBI group(P<0.05).Compared with TBI group,the expression of Bcl-xL was significantly decreased(P<0.05).However,there was no significant change in the expression of other apoptotic factor FasL,Bcd-2,Bax,caspase3.4.The expression of Bcl-xL,the recovery of nerve function and the inhibition of cell apoptosis were better than those of NSC transplantation 7 days after operation(P<0.05).Conclusion:1.Established the free-falling TBI model.2.Successed extraction and cultivation of NSCs.3.Successed constructed HSV-1 Bcl-xL ORF recombinant.4.NSCs transplantation and human HSV-1 transporter Bcl-xL-ORF group can promote the expression of Bcl-xL gene,reduce neuronal apoptosis,and promote the recovery of neural function after TBI,but the effect of human HSV-1 transporter Bcl-xL-ORF group is better than that of NSCs transplantation.