Lentiviral Vector Mediated Nurrl Overexpression Of Neural Stem Cells For The Transplantation Of Parkinson's Disease Rat

Objective:Nurrl gene can promote the differentiation of neural stem cells into dopaminergic neurons in vitro studies.entiviral vector mediated Nurrl overexpression of neural stem cells were established,further to study on the curative effect of Nurrl overexpression in rat model of neural stem cell transplantation for the treatment of Parkinson's disease and to explore its mechanism in vivo,and it must be a new way to the treatment of Parkinson's disease by cell transplantationMethods:1.The Nurrl gene was amplified by PCR,and then was conducted by double enzyme digestion with pLenO-DCE lentiviral vector,respectively.Purification of the digested products were connected or recombinant orientation,its products were transformed into bacterial competent cells,the clones were identified after PCR,sequence and analysis of steps to construct the recombinant lentiviral vector of Nurrl,and then transfected with 293T cells.The cell supernatant was collected after the concentration of virus titer was determined.2.The neural stem cells were isolated and cultured from 14 days of pregnancy fetal rat brain tissue in serum-free medium.The growth of the cells was observed every day,and then subcultured after 5-7d.Nestin immunofluorescence assay was used to verify the characteristics of stem cells,and the specific markers of beta III Tubulin,TH,and GFAP were used to identify the multiple differentiation potential.3.The multiplicity of infection was determined by the preliminary experiment of lentivirus infection,and the expression of Nurrl was detected by fluorescence,PCR and Western blot after the infection of NSC with MOI = 20.4.SD rats were given a intracerebral stereotaxic injection of 6-OHDA into rat medial forebrain bundle,ventral tegmental area and caudate putamen nucleus that dopamine neurons are densely populated.The average rotation speed of 30min was counted with apomorphine induced rotation test,and the PD rat model was identified.5.The successful PD rats were randomly divided into 3 groups:the control group was transplanted with equal volume of PBS to replace NSC;the NSC group was transplanted with empty lentiviral vector mediated NSC;Nurrl group was transplanted with the Nurrl recombinant lentiviral vector mediated NSC.The rats were tested for rotational behavior induced by intraperitoneal injection of apomorphine,respectively,after transplantation treatment 2 weeks,5 weeks,8 weeks.The expression of TH and DAT protein in brain was detected by Western blot at the same time point;The rat brains were taken at 8 weeks,then the brain morphology and TH expression were detected with immunohistochemical fluorescence;The therapeutic effect and its mechanism were compared and analyzed.Results:1.In this study,we successfully constructed the lentiviral vector pLenO-DCE-Nurrl expressing Nurrl gene.2.Primitive culture of the fetal rat midbrain cells suspension can be used to form hundreds of suspended neurospheres with a diameter of about 200 m around 7 d,which is positive by Nestin immunofluorescence assay.The beta-?-Tubulin,TH and GFAP positive cells were detected by inductive and differentiating culture.The results showed that neural stem cells had the multilineage ability to differentiate into dopaminergic neurons and astrocytes.3.Nurrl overexpression of primitive neural stem cells infected by lentiviral vector could express fluorescence after 48h.The fluorescence was most significant at the time of 5days.The negative control group and Nurrl group showed green fluorescence,and there was similar number of fluorescent cells in each field of vision,which indicated that the imported gene had made no difference on the transfection efficiency of plasmid.RT-PCR and Western blot could detect the transcription and expression of Nurrl in the cells.4.SD rats were induced by apomorphine test after given a intracerebral stereotaxic injection of 6-OHDA,which found that rats couldnot control themselves and continuously roated to damage contralateral.Starting from the second week,the number of roated rats was significantly increased and the rotation speed was faster than before.The trend of this slowed down after 4 weeks,and the rotation number and rotation speed of rats were relatively stable at the end of the 6 week after operation.Finally,43 rats were spin-stabilized and the average rotation speed was over 7rpm/min,which could be regarded as a successful PD model,the successful rate of PD was 58.1%5.The experimental rats were tested for rotational behavior induced by apomorphine injection after the transplantation.The results indicated that the rotational behavior of the PBS control group rats had no obvious improvement;Compared with the control group,the abnormal rotation number of the simple NSC group rats decreased from the beginning of the second week,the abnormal rotational behavior was improved to a certain extent;2 weeks after transplantation,the abnormal rotational behavior of the Nurrl group rats was more significantly improved than the NSC group along with time,but it still couldnot reach the normal level until 8 weeks.6.The immunofluorescence assay of TH showed that there were more transplanted cells survival in the Nurrl group than the simple NSC group.The Nurrl group had also a higher rate of differentiation,and the expression of GFP fluorescence was not less than 8 weeks in vivo.The transplantation of neural stem cell could promote the endogenous expression of TH positive cells.7.The experimental results of Western blot showed that the expression quantity of TH and DAT protein in the brain was significant higher in the Nurrl group than that of the NSC group,and increased gradually over time.The expression level of these two proteins was parallel with the improvement of the animal's behavior.Conclusions:In this study,we successfully constructed the lentiviral expression vector of Nurrl.And the Nurrl overexpression of neural stem cells intracerebral transplantation could significantly improve the effect of transplantation in the treatment of PD model rats in a certain period of time.Nurr1 can be used as an important target for cell transplantation therapy of PD,which provided experimental basis for clinical treatment.