Effect Of Bone Marrow-Derived Mesenchymal Stem Cells Transplantation On The Apoptosis And Expression Of P75NTR, TrkA In Perihematoma Tissue In Rats With Intracerebral Hemorrhage

Intracerebral hemorrhage(ICH) is a kind of serious cerebrovascular disease of high incidence, morbidity and mortality in central nervous system.It has been indicated that apoptosis of nerve cell in the tissue surround hematoma is the critical reason for the neurological dysfunction.P75neurotrophin receptor(P75NTR) and tyrosine kinase A(TrkA) receptor are both receptors of neurotrophin.They have antagonism effect in nerve growth factor(NGF) mediated neural cell protection. Recently, Bao G found in brain of patients with cerebral hemorrhage,the expression of P75NTR up-regulated, the expression of TrkA did not have a significant change, prompted that they both may participate in the secondary cell apoptosis in cerebral hemorrhage.Numbers of studies have shown that stem cell transplantation can improve the nerve function defect symptom in ICH, but there is no research which report the role of P75NTR with TrkA in Bone marrow mesenchymal stem cells(BMSCs) transplantation improving nerve function defect. BMSCs derived from the bone marrow,not involving ethics,no immune rejection reaction,can be self-donor transplanted.What’s more,they have multiple differentiation potential and a variety of roles of mechanism,easy to acquire prepare and preserve,not easy to malignant transformation therefore they have broader prospects for clinical applications.Whether transplantation of BMSCs play a role in anti-apoptosis in tissue around hematoma and by regulating the expression of P75NTR and TrkA to improve nerve function needs further research.This research adopted collagenase induction method to build ICH model,then stereotaxic injection of BMSCs to the tissue surround hematoma.The neurological function scores at different time points after surgery and the different expression levels of P75NTR, TrkA and the number of apoptotic cell in the tissues around hematoma in the sham group, model group and BMSCs group were observed in order to explore weather the transplanted BMSCs play an anti-apoptotic role by regulating the expression of P75NTR and TrkA to improve neurological function.So the mechanism how BMSCs playing a role in improve the function of ICH model can be better revealed,then give more support to the clinical application of BMSCs.Objectives:To explore whether transplantation of BMSCs play a role in anti-apoptosis in tissue around hematoma by regulating the expression of P75NTR and TrkA to improve nerve function.Methods:The clean male250-300g weighted Sprague-Dawley(SD) rats were randomly divided into sham group, ICH models group and BMSCs transplantation group.The Control was only stereotaxic punctured without injection of collagenase.The method of stereotaxic injection of collagenase into the striatum of rats was used to build rat ICH mode.Whole bone marrow repeated adherent method was adopted to separated and purified BMSCs,5th to8th generation of which then were transfected by lentivirus carrying Green fluorescence protein (Green fluorescence protein, GFP)gene.24h after model surgery, BMSCs group was stereotactic injected with2x105successfully transfected BMSCs suspension to the hematoma in rats.On day1,3,7,14after ICH modeling, neurological function were tested by mNSS and MLPT scales in all the groups.Then they all were sacrificed at day14then the rat brain were fixed, embeded and sectioned into slices observing the migration and survival of BMSCs.Immunohistochemical and western-blotting were adopted to detect the expression of p75NTR、TrkA in the tissues around hematoma,the terminal deoxynucleotidyl transferase-mediated dUTP nick end.labeling(TUNEL) immunofluorescent staining to detect the apoptosis cells,Results:1. Primary cultured BMSCs appeared like spindle and fusiform.After P2,they formed uniform spiral shaped. Flow cytometry was used to detect the purification of BMSCs.The positive rate of CD105and CD44was98.2%,99%, and the negative rate of CD34and CD45was100%,99.8%.It showed the purity is high. BMSCs after successful transfection appear green swirl under fluorescent microscope.2. After modeling, rats appeared obvious neurologic impairment, symptoms lasted for3to5days,after7d rats had different degrees of recovery. The sham group had no significant nerve dysfunction.The mNSS and MLPT scores of BMSCs group on7d,14d were significantly lower than ICH rats,the difference has statistically significance (P<0.05).3. BMSCs with green fluorescence protein can be seen clearly gathered around the hematoma, while only a few scattered distribution can be seen in the contralateral under fluorescence microscopy.4.14d after operation the expression of P75NTR around the hematoma in model group and BMSCs group were increased compared with sham group, while the one of BMSCs group decreased significantly in contrast with model group, the difference had statistical significance (P<0.05).5. The expression of TrkA in model group and sham group had no significant difference (P=0.117),that of BMSCs group had obvious rise compared with model group and control group (P<0.05).6. TUNEL positive cells around the hematoma of the model group and BMSCs group was higher than sham group and that of BMSCs group decreased significantly in contrast with model group, difference have statistical significance (P<0.05).The expression of P75NTR was positively correlated with the number of TUNEL positive cells (r=0.955, P<0.05).Conclusions:1. BMSCs which were stereotaxic administrated into cerebral hemorrhage rats model can survive and significantly enhance the recovery of neural function of intracerebral hemorrhage rat models.2. The expression of P75NTR in tissues of intracerebral hemorrhage rats rised and the apoptotic cells surrounding hematoma increased, while TrkA had no obvious change. P75NTR may mediate the cell apoptosis around the hematoma in cerebral hemorrhage rats.3. Transplantation of bone marrow mesenchymal stem cells can significantly down-regulated the expression of P75NTRs and raise the expression of TrkA around the hematoma to antagonismcell apoptosis around hematoma in cerebral hemorrhage rats.It may be one of its mechanism to promote neural functional recovery.