Effect Of CREB-BMSCs Transplantation On Ameliorating Learning And Memory Disorder In PD Model Rats

The cognitive disorder caused by Parkinson’s Disease(FD)is a typical non-motor symptom in clinical practice,among which learning and memory disorder is one of the significant manifestations.The cAMP Response Element Binding Protein(CREB)is a significant transcription factor in nucleus,which plays a key role in the regulation of learning and memory in the form of phosphorylation.Bone Marrow Mesenchymal Stem Cells(BMSCs)have been shown to improve motor dysfunction in PD model rats by intracerebral transplantation.On the basis of the study of the relationship between the development of learning and memory disorder in PD rats and the changes in the expression of p-CREB in the brain striatum,the BMSCs modified by CREB gene(CREB-BMSCs)were transplanted into the brain stria of PD model rats to study the effect of CREB-BMSCs transplantation on learning and memory disorder in PD model rats.1.Changes of learning and memory ability in PD model rats and p-CREB protein levels in the brain striatumWith the stereotaxic apparatus,PD rats were obtained by injecting 6-OHDA into the striatum.2 weeks later,rats were injected with APO to induce rotations and successful PD models were selected based on rotations ≥ 7r/min.After 2,5 and 8 weeks,Morris water,maze test was used to evaluate the learning and memory ability in PD model.The expression of p-CREB in the lateral brain striatum of PD model rats was detected by immunohistochemistry and western blot.After modeling for 5 and 8 weeks,the number of rotations in successful PD model rats was 9.13±0.62 r/min and 10.06±0.68 r/min,which was more than 7r/min,indicating that the PD models were steady.The results of Morris water maze test showed that the time of the PD model rats in the quadrant of the target platform was lower significantly than that of the normal rats(p<0.01,n=10).And the learning and memory ability of PD rats gradually declined.The analysis of Immunohistochemical showed that the number of p-CREB positive brown-stained cells in the striatum of PD rats gradually decreased with the progression of disease.The detection results of protein showed that the expression of p-CREB in the striatum decreased in PD rats,which is significantly lower than that in normal rats(p<0.01,n=5).The results suggest that the learning and memory abilities of PD rats attenuates gradually along with the progression of PD and the expression of p-CREB in the striatum also decreases.It indicates that the development of PD learning and memory disorder may be related to the decreased expression of p-CREB.2.CREB genetic modification of rats BMSCsThe recombinant eukaryotic expression vector pCDNA3.1(+)-CREB was transfected into BMSCs by Lipofectin 2000 to construct CREB-BMSCs.The expression of p-CREB in CREB-BMSCs was detected by immunofluorescence and western blot.CREB-BMSCs were induced to differentiate into neuron like cells in vitro by using p-mercaptoethanol,investigating the differentiation potential of CREB-BMSCs and the expression level of p-CREB after differentiation.The results of the enzyme digestion and sequencing of the recombinant vector showed that the recombinant vector of CREB was successfully constructed.The positive cells of FITC staining in the CREB-BMSCs group were significantly increased than BMSCs group,and the CREB-BMSCs can highly express p-CREB(p<0.01,n=5).After being induced to differentiate,CREB-BMSCs could specifically express three kinds of marker proteins NSE,Nestin and GFAP of neuronal cells.The results indicate that CREB-BMSCs still have the potential to differentiate into neuron like cells and can express p-CREB after differentiation.3.CREB Gene Modified BMSCs Transplantation for PD RatsThe fifth week after modeling,the experimental rats were divided randomly into PBS group,BMSCs group and CREB-BMSCs group.Rats in each group were injected with 10μL PBS,BMSCs(105/μL)and CREB-BMSCs(105/μL).After 2,5 and 8 weeks,the rats were induced to circulate by APO.The learning and memory ability in the rats was detected by Morris water maze test.Simultaneously,The expression of p-CREB in the lateral brain of rats was detected by immunohistochemical technique and western blot,and the improved effect of every group was analyzed.Compared with rats in PBS group,the number of rotating rings in BMSCs group and CREB-BMSCs group decreased gradually after transplantation.The number of rotating rings in CREB-BMSCs group was significantly lower than that of BMSCs group after 5 weeks(4.67±0.87r/min vs.5.73±0.77r/min,p<0.01,n=20).The results of Morris water maze test showed that the learning and memory disorder was improved and the learning and memory ability was also improved in BMSCs group.In the same time,the Amelioration of learning and memory disorder in CREB-BMSCs group was better than that of BMSCs group.The platform quadrant time of CREB-BMSCs group was significantly more than that of BMSCs group at 8 weeks(26.36±1.73s vs.19.34±1.51s,p<0.01,n=10).The results of immunohistochemical showed that the number of positive cells in the brain striatum of BMSCs group increased slowly.The number of positive cells in CREB-BMSCs group increased significantly than that of BMSCs group.The detection of protein showed that after 2,5 and 8 weeks,the expression of p-CREB in the brain striatum of CREB-BMSCs group was significantly higher than that in BMSCs group(p<0.01,n=5).The results suggest that the expression of p-CREB in the striatum of rats gradually increases and the learning and memory ability is increased by intracerebral transplantation of BMSCs and CREB-BMSCs.However,the effect of CREB-BMSCs group is better than BMSCs group.The results of this study suggest that the CREB-BMSCs transplantated into the brain of PD rats may migrate and differentiate into neuron like cells,ameliorating the impairment of PD and learning and memory disorders by increasing the expression of p-CREB.This study provides experimental basis for clinical gene therapy for PD dyskinesia and learning and memory disorder.