An Experimental Study Of Treatment Of A Rat Model Of Parkinson's Disease By Transplantation Of Neural Stem Cells Modified By Nurr1 Gene And Induced To Differentiate By All-trans Retinoic Acid/ginkgo Biloba

Background:Parkinson's disease(PD),characterized by the degeneration of dopaminergic neurons,is a common neurodegenerative disease mostly affecting middle-aged and old people.The key clinical features are tremor,stiffness,bradypraxia,gesture balance disturbance.At present clinical therapy of PD mainly depend on drugs and surgery,both of which are symptomatic treatment,consequently, the state of illness deteriorate progressively,a great many patients'symptom can not be controled effectively.NSCs are multi-directional differentiation potentiality neuro-precursor cells,can be differentiated into different neuronal cells or glial cell.NSCs are recogonized as a potential cell source for cell transplantion and gene therapy of PD.Nuclear related regulator-1(Nurr1),as a transcription factor of thyroid hormone/retinoic acid nuclear receptor superfamily,is specifically reguired for the induction and maintaining of dopaminergic neurons.Our previous work suggested that Nurr1 gene transfer combined with all-trans retinoic acid/ginkgo biloba (ATRA/Egb) could promote NSCs to differentiate into dopaminergic neurons.However,the therapeutic effects of neuronal cells produced bythis strategy is not proved.Objective:To observe the therapeutic effects of transplantion of neural stem cells co-induced to differentiate by Nurr1 gene transfer and all-trans retinoic acid/ginkgo biloba(ATRA/Egb) in vitro transplantation in a rat model of Parkinson'disease(PD).Methods:NSCs from brain of embryonic rats cultured in vitro,Nurr1 expressing plasmid(pcDNA3.1-hygro-Nurr1) was transfered into the NSCs by electroporation and the Nurr1 expressing NSCs were obtained by hygromycin selection,Nurr1 gene transffered NSCs were induced to differentiate by adding all-trans retinoic acid/ginkgo biloba(ATRA/Egb) into the medium and withdraw of EGF/bFGF for 1 week.then,the cells were collected and prepared as cell suspension for transplantation.In addition,NSCs transfferred with Nurr1 but not treated with ATRA/Egb.NSCs treated with ATRA/Egb but not transffered by Nurr1 gene and NSCs without any specific treatment were also allowed to differentiate,the cells suspensions were prepared the control groups.The 46 6-hydroxydopamine(6-OHDA)-induced hemiparkinsonian rats were allocated randomly into 4 groups.(1)Nurr1+ATRA/Egb group:receive grafting of NSCs-derived cells modified by Nurr1 gene and treated by ATRA/Egb;(2) ATRA/Egb group:receive grafting of NSCs-derived cells treated by ATRA/Egb but not transffered by Nurr1 gene;(3):Nurr1 group:receive grafting of NSCs-derived cells modified by Nurr1 gene but not treated with ATRA/Egb;(4)Nurr1 group:receive grafting of NSCs without any specific treatment.NSCs with brdu-labeled(2×105cells/each model) (NSCsgroup),NSCs induced by RA/Egb(RA/Egb group),NSCs modified by Nurr1 gene(Nurr1 group),NSCs modified and treated by RA/Egb(Nurr1+RA/Egb group) were transplantated into right striatum of PD rat respectively.The Apomorphine induced rotation test was taken from the firt week to the twelfth week after transplantation to observe to the behavior improvment.The rats were sacrificed and the brain were processed for immunohistochemical study.The brain sections were examined for the expression of brdu,nestin,Nurr1 and TH by immunohistochemical staining afer one week,two week,one,two,three months of NSCs transplantation,meanwhile determining survival,immigration and differentiation of NSCs in the brain of PD model after transplantion and counting the number of TH-positive cells to study the change of TH positive cells number in the brain.Results:The rotation behavior in all groups were significantly improved after transplantation,especially in Nurr1+RA/Egb group(P＜0.01),In immunohistochemical staining some TH-positive cells could be found in the implanted region of all groups.In Nurr1+RA/Egb group,the more TH-positive cells were found,neurite of TH-positive cells were more intensive,Some Nurr1-positive be found in the implanted region of Nurr1 group and Nurr1+RA/Egb group,but nestin-positive cells can not be found in the implanted regionConclusions:The transplantation of NSCs derived neuronal cells modified by Nurr1 gene and treated by all-trans retinoic acid/ginkgo biloba could improve the rotation behavior of rat model of PD.The implanted cells could survive, immigrate,differentiate in the brain of PD rats,some cells were found to express TH,Nurr1.These results indicate that neuronal cells derived from NSCs by Nurr1 gene modification and ATRA/Egb differentiation induction may be developed as an useful souce for cell transplantation therapy for PD.