| Parkinson's disease(PD) is a common central nervous system degenerative disease happened in middle and aged people.Transplantation of dopaminergic neuron is one of the effective measures in treating PD,which requires suitable and sufficient dopaminergic neuron cell supply.Because of the multiple differentiation potency and self-renewal ability, neural stem cells(NSCs)-highly nondifferentiated cell-are regarded as ideal donorcells in treating cerebral diseases by cell transplantation.However,normally only less than 0.01% of NSCs were differentiated into dopaminergic neuron cells.Therefore,in order to correct the symptoms of PD effectively,NSCs must be oriently differentiated into dopaminergic neuron cells before transplantation.Some studies found that the differentiation of NSCs was affected by many factors in the differentiation microenvironment,which included IL-1α,IL-11,Leukemia inhibitory factor(LIF),and Glial cell-derived neurotrophic factor(GDNF) currently.Stoch et.al found that by adding secretory fluid of striatum cells to the mixture of above-mentioned factors, The differentiation proportion of NSCs which were originatied from human mesencephalon into TH positive dopaminergic cells was increased by 6 times,and the differentiated cells demonstrated mature shape of dopaminergic neuron cells.Hitoo et.al showed that NSCs differentiated into dopaminergic neuron cells with much higher number,bigger cell body and longer overshoot if they were transplanted into the striatum of PD rats made by 6-OHDA injection.Furthermore,they confirmed that more GDNF and bFGF were secreted from the damaged striatium compared with the contralateral control striatum using antibody blocked technique.All these studies indicated that some factors which can induce oriented differentiation of NSCs into dopaminergic neurons exist in striatum,and if the substantia nigra pathway was damaged by 6-OHDA,the contents of those factors will increase or some new factors may be released.In this study,a mono-side PD model was made by injecting 6-OHDA to the striatum, then the expression difference of mRNAs between health-side and damaged side was compared to analyze the change of secretory factors from the striatum after 6-OHDA damage.A differentially expressed cDNA library was built based on the results,from which several genes were selected to investigate their influence on the differentiation and proliferation of NSCs.In the first part of this study,we tried to build a 6-OHDA induced PD rat model and evaluated it using ethological,high performance liquid chromatogram and electrochemical methods(HPLC-EC).The aim was to provide a stable and reliable model for further screening of genes which influences the pathological process of nigra-striatum.The results are as follows:1.After injecting of 6-OHDA to the striatum of rats,the ethological changes appeared in a gradual manner.Slow-rotation emerged two weeks after injection and reached 7 turns/min at the fourth week,then kept this condition till the 16th week.2.The contents of DA and DOPAC,the metabolite of DA,detected using HPLC-EC methods showed that in the 6-OHDA injected side nigra tissue,DA decreased by 67%,from 0.79nmol/l to 0.26nmol/l,compared with that of the contralateral side,and DOPAC decreased by 62.5%.In the striatum tissue DA decreased by 86%,and DOPAC by 94%.3.The TH immunohistochemistry results showed that TH positive neurons distributed in the nigra and ventro-tegmentum area,which was in big polygon or cone shape,and the kytoplasm was brown.The semi-quantitative statistical results showed that the TH positive substance demonstrated no significant change between left and right nigra in the saline treated rats,but changed significantly 4 weeks after 6-OHDA injection in the 6-OHDA treated rats(P＜0.05),and this difference became more significant after 6,8,12 and 16 weeks(P＜0.01).In the second part the expression change of mRNA between health side and damaged-side of striatum was compared using SSH technique,and in combination of molecular clone and northern blot techniques a variant cDNA library was built.6 up-regulated and 6 down-regulated gene clones were selected and functionally analyzed, and genes with definite function included TrfR,RILP,Slclal and Mtpn,Hnrpab,Fpgt, Cipar1,fibulin 5,Il22Rα2.In the third part of this study,a pEGFP-Slcla1 eukaryotic expression plasmid was constructed,and transfected to the glial cells cultured in vitro,then the cell sap was extracted to analyze its influence on the differentiation of C17.2 neuronal stem cells.The results were as follows:1.The recombinant pEGFP-Slcla1 plasmid was constructed successfully.2.The recombinant pEGFP-Slcla1 plasmid was transfected to the cultured glial cells successfully,because after transfection a strong green fluorescent protein was observed. Furthermore,the expression of Slcla1 mRNA was very high.3.The cell sap from glial cells transfected with recombinant pEGFP-Slcla1 could enhance the numbers of TH positive cells,but this showed no difference with natural differentiated cells.In comparison with the normal differentiated cells,adding cell sap and cytokine to the culture medium could increase TH positive numbers by 1.5 times.In conclusion,a host of mRNAs expressed differently between damaged striatum cells and normal striatum cells in the 6-OHDA induced unilateral PD rats.6 up-regulated and 6 down-regulated clones were confirmed by screening and analyzing the differential expression cDNA library which was constructed based on the first-part results,and the down-regulated gene-Slclal could enhance cytokine induced differentiation of C17.2 stem cells into dopaminergic neuron cells.Further studies are required to have a better understanding of the molecular modulating mechanism of the proliferation and differentiation of neuronal stem cells.
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