| Parkinson's disease (PD) is a neurodedenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra compacta (SN) and a severe decrease of dopamine (DA) concentration in the striatum, resulting in resting tremor, rigidity, bradykinesia and postural imbalance. Up to now, there is still no method that can prevent or halt the progress of this disorder. Gene therapy remains a promising but as yet theoretical approach to the treatment of PD. Current gene therapy models for PD are described in two parts: genetic transfer of the biosynthetic enzymes for dopamine synthesis (i.e. tyrosine hydroxylase, the key enzyme of dopamine synthesis, TH), and genetic transfer of the genes encoding several neurotrophic factors or other antiapoptotic factors (such as bcl-2, JIP-1 gene, etc.) for protection of dopaminergic neurons. Neurotrophic factors are known to play important roles in the development of neural tissue and protection of neurons against various insults. Glial cell line derived neurotrophic factor (GDNF) has been demonstrated to be a potent factor for protection of nigral dopaminergic neurons against toxin-induced degeneration in vivo. Furthermore, GDNF as well as its functional receptors have been found in the substantia nigra compacta, suggesting that this neuretrophic factor plays vital role in supporting the survival of dopaminergic neurons in vivo via an autocrine or a paracrine mechanism. Recombinant adeno-associated virus can mediate safe, integrated andlong-lasting transgene expression in the brain without any immunogenicity and pathogenicity, it seems the most promising viral vector for gene therapy in the central nervous system.Up to now, the transfer of single gene in vivo is a main research approach in all of the experiments on gene therapy of PD. It has some defects because it's impossibe to recover DA content and prevent or halt degeneration of nigral dopaminergic neurons at the same time by single gene transfer, either TH gene or GDNF gene. For this reason, combination of TH and GDNF gene therapy would be a logical approach to the treatment of PD. TH and GDNF genes are respectively introduced into the striatum and SNc in the lesioned side in parkinsonian ratsusing separate recombinant adeno-associated virus vectors (rAAV), and the coexpression of TH and GDNF maybe result in better behavioral recovery compared with TH or GDNF alone.The main results in this study showed as follows:Two rAAV expression plasmids were constructed to prepare for TH and GDNF gene transfer. One expresses human TH (pSNAV-TH). The other expresses human GDNF (pSNAV-GDNF). The genes are under the control of the potent cytomegalovirus immediate/early promoter. Stably transfect BHK cell line with pSNAV-GDNF and pSNAV-TH, respectively, and select the transfected BHK cell line with G418 and prepare the multiclonal packaging cell lines. The two packaging cell lines can express TH and GDNF efficiently, respectively. Amplify the multiclonal packaging cells and then superinfect them with rHSV-rap-cap to get rAAV-TH and rAAV-GDNF pseudovirus. Further concentrate and purify the crude products to get purified pseudovirus, the titer is about 10n-1012 particles/ml. Infect MN9D cells with the purified rAAV virus respectively, the results showed both rAAV-TH and rAAV-GDNF can significantly increased the dopamine production of MN9D cells while while a control vector, rAAV-GFP, cannot. It indicated all the vectors we got have clear biological activity of gene transfer.A partially lesioned PD rat model was made by unilateral medial forebrain bundle transection with a wire knife, resulting in progressive degeneration of dopaminergic neurons in the SN and abnormal ipsilateral rotational behavior of rats induced by amphetamine. HPLC showed DA content in the striatum reduced about 65.4% three months after the transection. rAAV-TH, rAAV-GDNF and the control virus which encoding green fluorescence protein gene (rAAV-GFP) was injected into the striatum or/and the SN of the PD rats, respective...
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