| Parkinson's disease(PD) is characterized by a very common degeneration of central nervous system(CNS). The pathologic feature is that the dopaminergic neurons appear degenerative or deadly,the vitality of tyrosine hydroxylase(TH) decreases in nigra of mesencephalon and the quantities of dopamine(DA) of striatum-nigra pathway reduce significantly,then the clinical symptom of tremor or trembling in hands,legs;bradykinesia or slowness of movement;and rigidity in hands,legs;bradylinesia or slowness of movement; and rigidity or stiffness of the limbs and trunk appear.Many methods have been used to treat the disease.But none of them is very efficient. Tranditionally, L-3,4-dihydorxyphenylalanine(L-dopa) is one of the important drugs to treat PD at present.But it's effects go through poor due to the continuous decrease of dopaminergic neurons and TH in the substantia nigra.In order to meliorate PD'disorders,it is necessary to increase the number of dopaminergic neuron that synthesize and deliver dopamine into the striatum as a neurotransmitter. Gene therapy approaches for the correction of the dopamine deficiency in Parkinson'disease may be the most efficient method at present.Neural stem cells(NSCs)are isolated from the mouse CNS at 1990s.NSCs is characterized by 1) undifferentiated feature,2)self-renewing capacity,3) multipotentiality. In the other words,NSCs can be passaged and cultured for a long time and proliferate in vitro.Induced by some factors,NSCs should differentiate into neuron,astrocyte and oligodendrocyte. The neurons differentiated from NSCs have the whole electronic physiological function andwill make synapses and integrate with host brain after intracerebral implantation. So are NSCs the ideal vehicle cells.In the present study,we have obtained efficient retroviral transduction of TH gene into proliferating NSCs in culture,and we report that NSCs engineered in this way can induce functional effects after intrastriatal transplantation in the rat Parkinson model.The main methods and result are results are as follows:1. The pN2ATH recombinant retroviral plasmids were transferred into PA317 cells with liposome.Resistant colonies were cloned and maintained under G418 selection(400μg/ml),then the virus supernatant were harvested.2. NSCs were isolated,identified and proliferated in vitro.3. The NSCs were incubated in N2ATH viral supernatant and selection under G418. Then the resistant colonies were achieved.The expression of TH was detected both in NSCs and the differentiated neurons and glias using immuno-fluorescent cell chemistry methods.4. NSCs and TH-transduced NSCs were transplantation in the head of the caudate-putamen ipsilateral to the 6-OHDA lesion of rats.At different time after transplantation the rats were tested for apomorphine-induced rotation.The rotation of rats that had received grafts of TH-transduced NSCs revealed decreasing apparently 8 weeks after transplantation compared with the control graft group and NSCs group.5. The DA and DOPAC levels in striatal areas of the PD rats receiving TH-transduced NSCs after transplantation were higher than that of receiving NSCs grafts and Saline grafts,but lower than that of normal rats.In conclusion,we isolated ,identified and proliferated NSCs in vitro and the N2ATH recombine retrovirus can be transferred into NSCs.TH gene is able to express in the NSCs and differentiated neuron and glial cells. When transplanted,the TH-transduced NSCs grafts provided partial amelioration of apomorphine-induced rotational asymmetry. The results clearly indicate thatTH-transduced NSCs capable of producing DA can survive and function in the brain of a rat model of PD. These data demonstrate that the TH-transduced NSCs might serve as a useful source of donor cells for the gene therapy of PD.
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