| The research of Parkinson’s disease(PD)at the moment has tended to focus on cognitive impairment as PD Nonmotor Dysfunction.PD in patients with cognitive impairment seriously influence their lives,and gradually become the focus of all circles.Extracellular-regulated protein kinase 1(ERK1)belonging to the MAPK family,which involved in regulating multiple signaling pathways associated with learning and memory,plays an important role on the formation of learning and memory that LTP regulated.Bone mesenchymal stem cells(BMSCs)has proven its effectiveness of transplantation in PD model rats are well treated.This experiment study on the relationship between cognitive impairment associated with Parkinson’s disease development and the expression of ERK1,on this basis,by transfecting ERK1 gene into BMSCs and transplantation in rat model of Parkinson’s disease,evaluate the effectiveness of ERK1-BMSCs Transplantation in treating PD and the cognitive impairment associated with Parkinson’s disease.1.levels of ERK1 in Parkinson’s disease model ratsUsing stereotaxic technique,unilateral cerebral striate body injection by 6-hydroxy dopamine(6-OHDA)to prepare Parkinson’s disease rat model,two weeks after building we injection of morphine on apomorphine(APO)by intraperitoneal to induced rat,screening successful model.In order to study the relationship of ERK1 and PD,testing the cognitive impairment in behavioral science experiment with model rats learning and memory ability,detecting the ERK1’s express the rule of change in experimental rat Parkinson’s disease in the substantia nigra striatum By immunohistochemistry,Western blot(WB)and Q-PCR method.Circle the experimental results show that the mode in 2 weeks,rotating laps gradually increase.To avoid the black experimental results showed that model rats in the bright box observation time percentage were significantly(p<0.05,n=15)is lower than the normal control group(99.22±1.40%)in 2 weeks,Shuttle box test,according to the results of model rats active shuttle number significantly(p<0.01,n=15)is lower than the normal control group(8.29± 2.98).Morris water maze experiment,according to the results of model rats platform quadrant time significantly(p<0.01,n=15)is lower than the normal control group(16.21±5.20 s).The immunohistochemical results showed that the model group rats brain nigra region of ERK1 positive brown stained cells count were significantly decrease,WB test,according to the results of model group rats striate ERK1 protein expression levels were significantly lower than control group(p<0.01,n=5),and a downward trend.Fluorescence quantitative PCR results showed that 2 weeks after building striate body rats ERK1 relative mRNA expression quantity of 0.79±0.08,significantly(P<0.05,n=5)lower than the normal control group.2.ERK1 genetic modification construction of BMSCsTo build pCDNA3.1(+)-ERK1 eukaryotic expression vector,use Lipo 2000 to transfection the BMSCs by recombinant eukaryotic expression plasmid,in order to obtain ERX1-BMSCs,The experimental group was divided into ERK1-BMSCs and BMSCs group.Through the WB,Q-PCR and immunocytochemistry method to detect ERK1 expression in BMSCs and BMSCs.In vitro,using β-mercaptoethanol ERK1-BMSCs directional sample cells induced to differentiate into neurons,explore ERK1 genetic modification after BMSCs differentiation potential and its ERX1 protein expression ability after differentiation.Enzyme electrophoresis of carrier recombination results showed that enzyme cut the stripe size is consistent with the theoretical value(1104 bp).Tapping the sequencing results with NCBI query to Wistar rats ERK1 gene sequences,the carrier to build success,Immunocytochemistry test,according to the results not only a small amount of ERK1 positive transfection of BMSCs,FITC fluorescence transfection ERK1 of BMSCs appear more positive number of ERK1 FITC fluorescence cells.WB test,according to the results of ERK1 protein expression in the ERK1-BMSCs significantly(p<0.01,n=5)higher than that of BMSCs,results suggest transfection ERK1 gene successful high expressed ERK1 protein in BMSCs,Q-PCR results showed that ERK1 mRNA expression of relative amount was 5.40±0.13 in ERKl-BMSCs,significantly(p<0.01,n=5)higher than that of BMSCs group(1±0.07).ERK1-BMSCs after β-mercaptoethanol induced differentiation of immune cells in vitro,the protein Nestin protein and NSE protein expression was positive,negative GFAP protein,without differentiation ERK1-BMSCs were negative.Detection of differentiation of ERK1-BMSCs and not differentiation ERK1-BMSCs in the expression of ERK1,immunocytochemistry test,according to the results of ERK1-BMSCs differentiation after processing.Results suggest that pCDNA3.1(+)-ERK1 eukaryotic expression vector to build success,and using liposome transfection ERK1 gene did not affect the BMSCs differentiation potential,and ERKl-BMSCs can still express high ERK1 protein after induced differentiation of BMSCs.3.ERK1 Gene Modified BMSCs Transplantation for PD RatsThe PD model rats were divided into PBS treat control group,BMSCs transplantation,ERK1-BMSCs transplantation treatment groups,respectively positioning injection PBS(10 μL),BMSCs(10 μL,1 × 105 cells/μL cells suspension)in three groups of animal brain striate body,ERK1 BMSCs.Groups of rats intraperitoneal injection respectively APO induction,detect changes in behavior after transplantation treatment,and use to avoid passive experiment,shuttle box test and Morris water maze behavioral experiments to detect the change of the treatment group rats learning and memory ability.And to the point in time,take out the rat striatum group,using the immune histochemical method,WB technology and Q-PCR to detect changes of ERK1 at the treatment of the rat’s striate body,analyzed each treatment group therapy effect and to explore its action mechanism.Circle experiment results show that the treatment 2 weeks,ERK1-BMSCs group turned around the number was 5.08±0.86 r/min,significantly(p<0.01,n=10)below the BMSCs group(7.75±1.83 r/min).Avoid passive,shuttle,Morris water maze experiment,according to the results of BMSCs and ERK1-BMSCs transplantation after 2 weeks,the percent of rat bright box observation time were 99.22±1.11%and 99.33±0.795%,2 weeks rats bright case observation time were significantly more than the PBS treatment group(p<0.01,n=10),BMSCs after 2 weeks treatment,the initiative to avoid significant number was 5.90±2.42 times more than the PBS false treatment group(p<0.05,n=10),ERK1-BMSCs treatment 2 weeks active escape times were 7.40±2.46 times,are significantly more than the PBS treatment group(p<0.01,n=10).BMSCs after 2 weeks treatment,platform quadrant of 13.28±6.97s significantly more than the PBS treatment group(p<0.05,n=10),ERK1-BMSCs treat platform quadrant 2 weeks significantly(p<0.05,n=10)more than the BMSCs treatment group was 7.27±6.52 s.For the study of BMSCs and ERKl-BMSCs transplantation in the treatment of the expression of ERK1 striatum of rats and the effect of Immunehistochemical method,through testing WesternBlot,Q-PCR to detect the expression of ERK1,Immunehistochemical method results showed that ERK1-BMSCs and BMSCs group rats’brain striatum ERK1 positive cells in the body for all with increasing treatment period(n=5).Q-PCR results showed that 2 weeks after treatment,ERKl-BMSCs and BMSCs in treatment group ERK1 mRNA relative expression were 2.76±0.06,1.21±0.15,significantly(p<0.01,n=5)higher than that of PBS control group(1±0.07),ERK1-BMSCs group were significantly higher than that of BMSCs group(p<0.01,n=5).WesternBlot results showed that the transplantation of 2 weeks,5 weeks,8 weeks,the ERK1 protein expression of rats in ERK1-BMSCs group were significantly higher than that of BMSCs group(p<0.01,n=5),and in the treatment of 8 weeks after ERK1-BMSCs group,the ERK1 protein expression quantity has reached even beyond normal group,consistent with the Q-PCR results.Experimental results indicated that after ERK1-BMSCs and BMSCs transplantation in the treatment of PD model rats,the behavior and learning and memory ability,PD and its cognitive impairment has been improved,and ERK1-BMSCs transplantation treatment efficiency is better than single BMSCs transplantation therapy. |
