【作者】 柴怡；

【导师】 胡刚； 李皓；

【作者基本信息】 南京医科大学 ， 神经生物学， 2005， 硕士

【摘要】 ATP敏感性钾通道开放剂埃他卡林对H₂O₂诱导 PC12细胞损伤的保护作用及其机制的研究 帕金森病（Parkinson’s disease,PD）是好发于中老年人的慢性中枢神经系统退行性疾病,严重影响中老年人的生活质量和健康水平。PD的主要病理学改变是选择性黑质-纹状体通路的多巴胺（Dopamine,DA）能神经元退行性变、Lewy’s小体的出现以及纹状体DA含量的绝对减少。然而,对于导致DA神经元变性、坏死的确切机制目前还不清楚,临床也缺乏理想的治疗药物及手段。因此,探索PD的病因和研发富有潜力的治疗新药成为目前神经科学研究领域的热点之一。许多研究表明在PD病变过程中,黑质致密部DA能神经无线粒体呼吸链复合物Ⅰ活性下降,ATP合成减少,造成细胞能量代谢障碍;而ATP敏感性钾通道(ATP-sensitive potassium channels,K_ATP)是一类由胞内ATP/ADP水平调控、非电压依赖性的、直接耦联细胞代谢和电活动的特殊钾离子通道。包括本实验室在内的许多研究都已表明K_ATP在PD病变过程中发挥重要作用。 埃他卡林（Iptakalim,IPT）是由我国学者自行设计和合成的一个脂肪仲胺类小分子化合物,药理学、电生理学、生化研究和受体结合实验已经确认IPT具有K_ATP开放剂的特征且具有能够透过血脑屏障的独特优势。本实验室的前期研究工作发现IPT能够拮抗6-OHDA、MPP⁺、Rotenone等多种神经毒素导致的在体或离体细胞损伤。但是,IPT是否通过作用于PC12细胞上的K_ATP而实现保护作用?目前尚缺乏直接的实验证据,IPT神经保护作用的确切机制也有待于深入研究。 本文首先鉴定了PC12细胞上K_ATP亚基的组成,为IPT作用靶点更多还原

【Abstract】 Parkinson’s disease （PD） is one of the most common neurodegenerative disorders, affecting almost 1% of the population over 65 years old. The pathological hallmarks and biochemical changes of PD are the loss of nigrostriatal dopaminergic neurons, the reduction of dopamine content in the striatum and the presence of intraneuronal proteinacious cytoplasmic inclusions, termed "Lewy’s Bodies". Until now, the underlying mechanisms for the loss of dopaminergic neurons remain unclear although both genetic mutations and environmental factors have been identified as contributing to certain forms of this disorder. So there is lack of relatively good symptomatic therapy and medicine for PD. Hence, many studies are focused on searching for new potential therapeutic targets and agents for the treatment of PD. Recent evidence suggests that mitochondrial damage, particular to the respiratory chain of complex I （CXI, NADH/ubiquinone oxido reductase） resulting in oxidative and nitrative stress, underlies the pathology of PD. K_atp channels are activated rapidly in response to the decrease of intracellular ATP/ADP ration, coupling cell metabonism and its electrical activity. So many studies revealed that ATP sensitive potassium channels related to PD.We developed a new compound iptakalim, which has beendemonstrated to be a novel K_atp channel opener by pharmacological, electrophysiological, and biochemical studies. Notably, IPT is one of the KCOs that can pass through blood-brain-barrier. Our previous research have revealed that IPT could antagonize the toxicity of 6-OHDA, MPP⁺, Retonone. So in the base of this we identified the expression of subunits of K_atp channel on PC12 cells and established the model of oxidative stress to reveal the protction of IPT on oxidative injuried of PC12 cells and investigated possible pharmacological mechanism in the present study.1. The expression of subunits of K_atp channel on PC12 cellsTo determine whether SUR and Kir subunits are present in PC 12 cells, we used specific antibodies against SUR1, SUR2, Kir6.1 and Kir6.2 in Western blotting analysis and immunocytochemistry. The mRNA expression of subunits in PC 12 cells were determined by RT-PCR. PC 12 cells expressed mRNA for Kir6.1, SUR2 subunits of K_atp channels. Western blotting and immunocytochemistry of PC12 cells revealed a same results, respectively.2. Effects of iptakalim on H₂O₂ induced cytotoxicity in PC 12 cellsNeurotoxity injury was induced by H₂O₂ in PC12 cells. The cell viablity was tested by MTT assay. DPT markedly mitigated H₂O₂-induced decrease of cell survival and morphological alteration in PC12 cells. But 5-HD can abolish part of protective effect of IPT.3. Mechanisms of iptakalim aganinst H₂O₂ induced neurotoxity.The glutamate release from PC12 cells was measured by HPLCcombined with fluorescent detector analysis. 10 uM IPT or 100 uM diazoxide can inhibit 40 uM H2O2 induced glutamate release increasing. Changes in the intracellular free Ca2+ concentration （[Ca2+]j） were determined in fluo-3 AM loaded PC 12 cells. The increase of [Ca2+]i induced by H2O2 can be prevented by IPT. Hoechst 33342 staining results showed that some of the H2O2-treated cell exhibited highly condensed and fragmented nuclei morphology, which were the typical characteristics of apoptosis. In contrast, the cells in the culture with 10 uM IPT or 100 uM diazoxide showed normal cell nuclei morphology. If the cells were pretreated with 10 uM IPT and 100 uM diazoxide, 40 uM H2O2-induced over-expression of Bax and lowered Bcl-2 levels were attenuated further suggesting a possible link between IPT’s anti-apoptotic role and gene modulation. But the cells exposed with 5-HD together can partly abolish the effect of IPT.Conclusions:1. PC12 cells expressed mRNA and proteins for Kir6.1 and SUR2.2. IPT provided neuroprotection against H2O2 induced cytoxixity by activating mitochondrial KAtp channels decreseing the release of glutamate and intracellular free Ca2+ concentration.3. IPT exerted certain protective更多还原