The Effects Of Exogenous Dopamine On Over-expression Of α-Synuclein In Dopaminergic Neuron

Objectives To approach the cell functional effects of exogenous dopamine onover-expression ofα-Synuclein in dopaminergic neuron and provide vitroexperimental evidences to elucidate the puzzle about"Does L-dopa/DA aggravate thepathogenetic condition of Parkinson disease?"Methods (1) Constructing vitro cell model of PD:Dopaminergic cell lineSK-N-SH was transfected with recombinant plasmidα-Synuclein-pcDNA3.1 byliposome.The stable clones were gained through screening of G418 and the levels oftranscription and expression about destinational geneα-Synuclein were identified bymeans of RT-PCR and Western blot.(2) Observing the cell functional effects ofover-expression ofα-Synuclein on cell line SK-N-SH:Defined cell strains weretransfected withα-Synuclein-pcDNA3.1 and pcDNA3.1 as experimental group andcontrol group respectively.By means of MTT,Chromatometry,Flow cytometry,RT-PCR and Western blot,the cell growth curve and activity,contents of MDA,earlyapoptosis rates,levels of transcription about the gene Caspase-3,P53,Bcl-2,Bax,SOD 1,SOD2 and CAT,which were related with apoptosis and oxidative stress werecontrasted,and levels of expression about Caspase-3,P53,Bcl-2 and Bax were alsobe evaluated in both groups.(3) Observing the cell functional effects of exogenousdopamine on over-expression ofα-Synuclein in cell strain SK-N-SH:Cell straincontaining none DA was defined as control group and experimental groups into 50uM DA group,100 uM DA group,300 uM DA group and 500 uM DA group by thefinal concentration of exogenous DA were classified.By means of MTT,Chromatometry,Flow cytometry,RT-PCR and Western blot,the cell activity,contentsof MDA,early apoptosis rates,levels of transcription about the gene Caspase-3,P53,Bcl-2,Bax,SOD1,SOD2 and CAT and levels of expression about Caspase-3,P53,Bcl-2 and Bax were detected in different groups respectively.Results (1) Construction of vitro cell model of PD:SK-N-SH cell line wassuccessfully transfected with recombinant plasmidα-Synuclein-pcDNA3.1 andpositive clones was gained by screening with G418 350ug/ml.Outcomes of RT-PCR and Western blot manifested that the transcriptional and expressional levels aboutα-Synuclein in cells transfected withα-Synuclein-pcDNA3.1 were higher than thosetransfected with pcDNA3.1.It showed that the cell model over-expressing wild-typeα-Synuclein was successfully constructed.(2) Ceil functional effects ofover-expression ofα-Synuclein on SK-N-SH:Outcomes of MTT showed thatexperimental cells growed slower and had less activity than control cells.Outcomesof chromatometry showed the content of experimental cell homogenate MDA washigher than that of control group.Results of flow cytometry showed the earlyapoptosis rate of experimental cell was higher than that of control group.RT-PCRresults showed the transcriptional levels of Caspase-3,P53,Bax,SOD1,SOD2 andCAT in experimental group were higher than that of control group,but Bcl-2 inexperimental group was lower than that of control group.Outcomes of Western blotalso showed that the expressional levels of Caspase-3,P53 and Bax in experimentalgroup were higher than that of control group wherase Bcl-2 in experimental groupwas lower than that of control group.(3) Cell functional effects of exogenousdopamine on over-expression ofα-Synuclein in SK-N-SH:Outcomes of MTT,Chromatometry,Flow cytometry,RT-PCR and Western blot showed respectively thatcells activity,contents of cell homogenate MDA,early cell apoptosis rates,transcriptional levels about Caspase-3,P53,Bax,SOD1,SOD2 and CAT and theexpressional levels about Caspase-3,P53 and Bax increased progressively followingwith the augmentation of exogenous DA concentration,but those of Bcl-2 decreasedprogressively.Conclusions (1) Over-expression wild-typeα-Synuclein enhanced the level ofoxidative stress in cell line SK-N-SH and accelerated cell apoptosis.(2) ExogenousDA also enhanced the level of oxidative stress in SK-N-SH cells which was overexpressedα-Synuclein and accelerated cell apoptosis and this effect was dosedependent.(3) Our vitro experiments implied that L-dopa/DA could aggravate thepathogenetic condition of PD by toxic to survivally dopaminergic neuron insubstantia nigra accompanying with the improvement PD patients' motor symptoms.