Tyrosine Hydroxylase Gene Transfection Of Neural Stem Cells And Expression

The Transfection and Expression of TyrosineHydroxylase in Neural Stem CellsAbstractThe progressive loss of dopaminergic cells and a deficiency of tyrosine hydroxylase (TH) in substantia nigra are the major pathological characteristics of Parkinson's disease (PD), resulting in the decreasing of dopamine (DA) in striatum and the complex symptoms. Traditionally, L-3.4-dihy droxyphenylalanine (L-dopa) is one of the important drugs to treat PD at present But their effects go through poor due to the continuous decrease of dopaminergic neurons and T.H in the substantia nigra. In order to meliorate PD's disorders, it is necessary to increase the number of dopaminergic neurons that synthesize and deliver dopamine into the striaturn as a neurotransmitter.Intracerebral transplantation of dopa.minegic tissues is now developing into a new measure to correct the symptoms of PD. It is well known that the symptoms of PD have been obviously improved following the transplantation of fetal mesencephalic cell suspension into the host striatum. Unfortunately, some factors limit their wide application in the clinic. For example, it is very difficult to obtain several embryonic brains with similar age at a short time for transplantation and intense ethical argument embarrasses the works. Some researches reported that the transplatated genetically engineered fibroblast and myoblast could be used to express TH in the host brain for the gene therapy of PD. But the long-term effects were also suspended because these cells were not able to survive in the host brain for a long time. For the above reasons, it is imperious to look for one kind of cell resources for the transplantation. Therefore, the cells must meet the following standards: 1. to be easily harvested and genetically engineered andto be cultUred and passaged fOr a long tiIne iK vitrO. 2. to survive and makeWses with the hoSt bthe de twlantatioirResearCers found the neural stem cells (NSCs) from the mouse centralnervous system(CNS) at l990s. NSCs is characterized by l).undifferentiated featUrs, 2). self renewing caPacity and 3). multipotentialit}LIn the other words, NSCs can be passaged and cultUred for a long tdrie andproliferate in vitrO. hidueed by some factors, NSCs should differentiate intoneuron, rtocyte and ollgodendrocyte. The neurons differentiated fromNSCs have the Whole electronic physiological function and will makesynaPses and integrat wt hoSt brain after intrcerebral imPlanation. Soare NSCs the ideai vehicle cells. In this stUdy, we constructed THcDNA witha deficient recombined retroviral veetor N2A and developed TH expressingplasndds. The plasndds were transferred into packaging PA3l7 cells by usingeleCtfOPoration and liPosome, then, the vha suPernan were harveSted.NSCs from El6 WistaI rat wer iso1ated, Prolifhaed and then the NSCs wasirifected using recombined retroviruses obtained from the PA317 cells. Theexpression of rn was detected both in the differentiated neurons and gliasusing driuno-fluorescent cell chendm methods. The results in this StUdywere summaried as fOllows:l. The rn-N2A plasndds were counCted and eXPanded.2. NSCs were iso1ated, identified and proliferated in viho.3. The oPtional conditions and methods fOr TH transferring into NSCs wereexplored.4. NSCs were induced by using bFGF and 5% FBS to differentiate intoneuroll, aStrOcyte and oligodendroCyte in vitro*5. The exPression of m were detected in the neurons and glia differentiatedfrom the NSCs which contained rnN2A.In one word, we isolated. identified and proliferated NSCs in vitro and themrecombined retrovirus can be transferred into NSCs. Also, TH gene is able to express in the differentiated NSCs. We confidently consider that the NSCs genetically engineered with TH can be used as a resource of donor cells for the gene therapy of PD.