| Parkinson’s disease is currently the world’s most common neurodegenerative movement disorder, and about2%of the world’s elderly people over the age of60are afflicted with the disease. Pathology, the most important feature of patients is the reduction of dopaminergic neurons of the substantial nigra pars compacta,which lead to the decrease of dopamine (DA) in the substantia nigra striatal pathway, resulting in a significant reduction in striatal DA content. The etiology and pathogenesis of Parkinson’s disease is not clear nowadays. Because of the complexity and diversity of the pathogenesis of PD, any drugs could not eventually slow down and stop or even reverse the degenerative process of patients’substantia nigra dopaminergic neurons and thereby restore the normal function of nigrostriatal pathway. Drug treatment, surgical treatment, or nerve cell transplant treatment can effect a radical cure, and they will also bring significant side effects.The lesion of Parkinson’s disease are mainly due to the decline in the level of dopamine in the striatum,which is caused by the degeneration of dopaminergic neurons, However, because of the part of the brain lesions in patients with Parkinson’s disease is small and easy to conduct the topical treatment, Parkinson’s disease has always been a popular nervous system diseases focusing on gene therapy research.In this paper, we adopt the enzyme-produced genes and Protective gene to make recombinant viral vectors can have the function of restoring dopamine level and protecting damaged cells,and then treated rat model of PD in order to achieve the best therapeutic effect.First, we choose glial cell line-derived neurotrophic factor (GDNF), which not only can protect nerve cells against the injury, but also have the ability to recover the damaged cells. Additionally, we infuse the three enzyme-produced genes: Aromatic-L-Amino-Acid Decarboxylase (AADC), Tyrosine Hydroxylase (TH), GTP Cyclohydrolase I(GCHI),which are necessary to produce dopamine, into striatal cells to replace damaged nigrostriatal system to produce dopamine and function normally. Therefore, the choice of these four genes, can fundamentally restore and protect the nigrostriatal system to make the Parkinson’s symptoms alleviated or effect a radical cure.In the chosen viral vectors for gene therapy, Adeno-associated virus is the most effective and safest virus for gene therapy of Parkinson’s currently. Until now,12species of AAV serotypes of human has been found. AAV2is the most extensive clinical research in gene therapy, but the infection rate for nerve tissue is low, and85%of the normal population has antibodies against AAV2. So rare AAVs gradually attract people’s attention. The AAV8used in this article is the most infectious viral vector found for nigrostriatal system. At the same time, the CAG promoter, as compared with the general promoter, has the ability of dirving the genes expressing efficiently and long-termly in the central nervous system.In this article, firstly we connected the tTH and GCH I through IRES,which can make tTH and GCH I play together to generate L-Dopa (the precursor of Dopamine).Secondly, because of the length of the therapeutic gene, we packaged GDNF and AADC respectively, and then generated three recombinant AAV virus-AAV8-CAG-GDNF, AAV8-CAG-AADC and AAV8-CAG-TTH-IRES-GCH I.In vitro, we used Cos-7cells to validate the production of dopamine through AADC, tTH and GCH I.At the same time, we confirmed that the virus-AAV8-CAG-zsGREEN have a good effect in infecting primary fetal rat striatal cells. Next, we prepared the6-OHDA Impairment Rat Model of Parkinson’s Disease,the success rate of which can get50%.Then we injected the three recombinant viruses as the pre-designed group into the striatum of rat model and evaluate therapeutic effect from behavioristics and immunohistochemistry. The results showed that, compared with the blank group, the rotation of GDNF group, enzyme-produced group and combination group decreased to approximately23%、37%、47%respectively,which confirmed that the combination of GDNF and enzyme-produced genes was the best treatment than any single group.Second, the frozen sections and immunohistochemistry of striatum in every experimental group showed the target genes that recombinant virus carried has a better expression, and the infested area can reach almost80%of the corpus striatum. So the design of this experiment has achieved the intended purpose, and laid the foundation for the gene therapy of the Parkinson’s disease. |
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