Construction Of Overexpressed DDC,TH And GCH1 Recombinant Lentivirus Vector And It’s Therapeutic Effect In 6-OHDA-induced PD Rat Model

Parkinson’s disease（PD）is a common and complex neurodegenerative disease in the world.It’s characterized by the progressive loss of dopaminergic neuron in substantia nigra pars compacta（Snc）,resulting in a decrease of dopamine（DA）,especially in the nigrostriatal pathway.The etiology and pathogenesis of PD are still unknown,which is not caused by a single factor,but the result of interaction with age,environment and heredity.Currently,no drug in clinical can slow down,prevent or reverse the process of dopaminergic neurons degeneration.Whether drug or surgery can not cure PD fundamentally,and these therapeutic schedules will cause drug-induced or surgery-induced adverse reaction.PD is the hot target disease of gene therapy,because the main pathological changes is the loss of dopaminergic neuron in Snc and the diseased region is small and definite.In this article,we constructed overexpressed dopamine decarboxylase（DDC）,tyrosine hydroxylase（TH）and GTP cyclihydrolase 1（GCH1）positive recombinant lentivirus（rLV）.Stereoscopic injection into the striatum of 6-OHDA PD rat models with DDC+TH+GCH1-rLV was performed.Then we observed the expression of virus and the change of rotation behaviors in animal models.We used the enzyme-synthesis gene recombinant viral vector for treatment in animal models,aimed to reconstruct the DA level in striatum and expected the desired therapeutic effect.Part I Construction and identification of DDC,TH and GCH1 rLV and control rLVObjective:To construct DDC+TH+GCH1 rLV and control rLV Methods:The target gene fragments DDC,SV40 early promoter,TH,mPGK promoter,GCH1wereamplifiedbyPCR,thenthepMT174vector（pLV-RSV-CMV-WPRE）was digested by BamHI-HF and XhoI endonucleases,and recycle carrier fragments for standby application.The homologous recombination of the target segments and the recycled linear expression vectors wererecombined under the action of the seamless reaction fluid.Then the transformants were sequenced after identification using colony PCR.If the results of sequencing were right,then we started to plasmid extraction.Finally the target plasmids and helper plasmids pCMV-dR8.9、pCMV-VSV-G was co-transfected into HEK293 cells for virus packaging.Then the virus particles were purified and concentrated for standby application.The sample was titrated by RT-PCR.DDC+TH+GCH1-rLV and control rLV were taken out and melt on theice,then infecting HEK293T cells.After 72 hours,we extracted the total protein of the infected cells and use the method of Western blot to detect DDC,TH and GCH1.Results:PCR results showed that specific DDC,SV40 early promoter,TH,mPGK promoter,GCH1 bands were found at about 1429 bp、412 bp、1614 bp、551 bp、791bp.We recovered 6508 bp linearized vector after using BamHI-HF and XhoI endonucleases to digest the pMT174 vector.The results of colony PCR showed that the positive plasmid was 1436 bp.In addition,the result of sequencing were right.Cotransfect HEK293T cells with helper plasmids.DDC+TH+GCH1-rLV and control rLV particles were obtained after 72 h,and their titers were 5.56×10⁸ v·g·mL^-1,1.07×10⁹ v·g·m L^-11 respectively.Lastly,the virus particles were packaged and stored at-80℃.The result of Western blot showed,in DDC+TH+GCH1-rLV infected team,the palce of DDC,TH,GCH1 protein molecular weight can see specific stripes expressed.Conclusion:DDC+TH+GCH1-rLV and control rLV has been successfully constructed.What’s more,their titer and the function of carried genes can meet the requirements of subsequent experiments.Part II The therapeutic effect of DDC+TH+GCH1-rLV in 6-OHDA-induced PD rat modelObjective:To evaluate the therapeutic effect of DDC+TH+GCH1-rLV in 6-OHDA-induced PD rat model and test the expression of target genesMethods:6-OHDA was stereoscopically injected into medical forebrain bundles（MFB）of rats at the two coordinates to construct unilateral complete destructed PD rat model.The standard of complete destructed PD rat model is that the number of rotation laps per minute is greater than 7or the number of rotation laps of 30 minutes is grater than 210.Through the method of Snc TH immunofluorescence staining,the damage of dopaminergic neurons in PD rat model was detected.24 successful PD rat models produced by 6-OHDA injection were divided into three groups randomly:normal saline group（n=6）,control rLV group（n=6）and DDC+TH+GCH1-rLV group（n=12）.Stereoscopic injectction into the Striatum of 6-OHDA-induced PD rat models with normal saline,control rLV and DDC+TH+GCH1-rLV respectively.Through the method of Striatum TH immunofluorescence staining,the positive rLV transferred cells in Striatum was detected.The levels of Dopamine and its metabolites of Striatum were measured by high performance liquid chromatography combined with tandem mass spectrometry（HPLC-MS/MS）in each group.Results:We stereoscopically injected 6-OHDA into medical forebrain bundles（MFB）of rats at the two coordinates to construct unilateral complete destructed PD rat model.According to the standard of rotation behavior,PD rat models were selected.After the Striatum injection of PD rat model,DDC+TH+GCH1-rLV group showed obvious rotation behavior significance in contrast with normal saline group and control rLV group（P<0.01）.DDC+TH+GCH1-rLV group presented prominent rotation behavior improvementt.In addition,with TH immunofluorescence staining,striatum of positive rLV transferred group can detect TH expressing positive cells.Compared with normal saline group and control rLV group,DA and its metabolites in positive rLV group appeared prominent progress（P<0.01）.Conclusion:DDC+TH+GCH1-rLV group showed significant behavior improvement,increased DA level of Striatum and stable gene expression.