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Studies On The Mechanism Of Dopaminergic Cell Death Induced By Oxidative Stress

Posted on:2003-10-28Degree:DoctorType:Dissertation
Country:ChinaCandidate:Q Z FuFull Text:PDF
GTID:1100360092965528Subject:Cell biology
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The pathological character of Parkinson's Disease (PD) is the loss of dopaminergic neuron in substantia nigra. The etiology of neuronal death in PD remains elusive. The environmental factors are becoming more and more emphasized. MPP+, the reacitive metabolite of l-methyl-4-phenyl-l,2,3,6-tetrahydropypridine (MPTP), one of the envirnment factors, is the inhibitor of mitochondrial complex I . It's toxic effect has relationship with reactive oxygen spacies (ROS). Dopamine (DA) is a natural neurotransmitter of the brain. It can be auto-oxidated into 6-hydroxydopamine (6-OHDA) and neuromelanin, so it is a strong oxidant. Hydrogen peroxide is belongs to a kind of ROS. So all the compound above can be used to produce oxidative stress. Alpha-synuclein is a member of a-synuclein family. Its importance in PD pathogenesis is highlighted in recent years, but the researching result is conflicting. Our study is to investigate the changes of a-synuclein in different time points by using the oxidative stress modal induced by these three PD-related toxins. Whether the mechanism of dopamergic cell of PD is apoptosis or nacrosis is not clear. The researching result of this field is conflicting in these years. Some researchers consider it as apoptosis, while some necropsy results don't support the hypothesis. Our study is to investigate the protein expression and biochemistry changes under oxidative stress to determine if apoptosis happens.Part â…  Establishment of the Oxidative Stress on Dopaminergic Cell ModelA dopaminergic neuroblastoma MES23.5 was derived using somatic cell fusion of rat embryonic mesencephalon cells and the murine neuroblastoma-glioma cell lineN18TG2. The cells were treated with MPP+ (100~800uM), H2O2(200~800uM), DA(50~800uM) and incubated for 24~72hrs. The cellular viability was measured using MTT assay. Result: MPP+ and DA in low concentration did not effect the cellular viability significantly while the cellular viability was significantly decreased under the higher concentration in a concentration and time dependent manner. The result suggests that MPP+ and DA in lower concentration have not any toxicity to cell even if exposed for a long time. Once the concentration reach a threshold value, the toxicity become irreversible. The H2O2 didn't have the threshold effect. Its toxicity to cell is concentration and time dependent. We chose MPP+200uM, DA 150uM and H2O2 200uM as the toxin concentration for the further researching.Part â…¡ The Effect of Toxin on the Marker of Cellular Oxidative StressThe cells were exposed to three kinds of toxin for 6h, 12h, 24h and 48h respectively. The free radical OH" and superoxide dismutase (SOD) were measured by chromatometry using the assay kit. The fluorescent probe DCFH2-DA and Rhodaminel23 were used for the cellular ROS and mitochondrial membrane potential ( A WM) respectively. The OH began to increase when the cells were treated with toxin for 6h. With the time prolongation, the OH" was increased gradually. The SOD was decreased only while treated for 48h. Our result suggests that MPP+, H2O2, DA can allinduce oxidative stress in time-dependant manner while the damage to anti-oxidant defence system only happened in the late phase.Part â…¢ The Effect of Oxidative Stress on a-synucleinExpressionOur study is to investigate the change of a-synuclein expression under the oxidative stress to understand the relationship among toxin, oxidative stress and a-synuclien expression. Result: Western blot display the increasing of a-synuclein expression in 24h and 48h, and the expression is higher in 48h than in 24h. The double-immunocytochemistry and laser scanning confocal microscopy showed a-synuclein immunostaining is stronger in toxin treated cells in general. In MPP+ and DA treated cells, the a-synuclein-positive staining was located in cytoplasm and were more intense in nuclei. In the cells of H2O2 treatment, a-synuclein immunostaining associated with cytoplasm and a-synuclein posistive granulae, which were TH imm...
Keywords/Search Tags:MPP~+, H2O2, dopamine, oxidative stress, free radical, reactive oxygen species(ROS), MES 23.5 cell, @-synuclein, tyroxine hydroxylase (TH), caspase-3
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