组蛋白去乙酰化酶抑制剂对帕金森病救援作用机制研究

发布时间：2018-06-23 18:40

本文选题：帕金森病 + 组蛋白去乙酰化酶抑制剂　；参考：《新乡医学院》2014年硕士论文

【摘要】：背景帕金森病(Parkinson's disease, PD)是第二大常见的神经退行性疾病,以黑质纹状体多巴胺含量减少,中脑黑质多巴胺神经元缺失为主要特征。目前,帕金森病的治疗主要是维持胆碱能和多巴胺能的平衡,缓解运动症状,尚没有治愈的方法。氧化应激和凋亡学说是帕金森发病机制中最重要的学说,有研究表明PD病人的黑质总是处于一种高度氧化应激状态。表观遗传机制组蛋白乙酰化和去乙酰化在帕金森病和其他神经退行性疾病中起着非常重要的作用,组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor, HDACI)对神经退行性疾病具有一定的保护作用。金属硫蛋白(Metallothionein2,MT2)是一种氧化应激抵抗因子,组蛋白去乙酰化酶抑制剂能上调MT2基因表达。多巴胺转运体(dopamine transporter, DAT)和囊泡转运体(vesicular monoamine transporter, VMAT2)在维持中枢神经系统的多巴胺等单胺类物质平衡中发挥着十分重要的作用。DAT和VMAT2启动子区CpG岛甲基化程度具有很大变异,从2%到90%,并与基因表达相关,显示表观遗传调控是DAT和VMAT2表达调控的重要机制。因此,组蛋白去乙酰化酶抑制剂是否通过上调MT2、DAT和VMAT2的表达进而对帕金森病产生保护作用,其表达变化是否通过表观遗传机制实现?这些有待与我们进一步研究。目的探讨组蛋白去乙酰化酶抑制剂对帕金森病细胞模型的保护作用及其可能的机制方法研究对象：多巴胺能神经细胞实验分组：对照组(DMSO)、模型组(0.5μM鱼藤酮、1mM MPP+,24h)丁酸钠干预组(1mM、2mM、5mM,48h) 1.采用四甲基偶氮哗盐(MTT)比色法检测细胞活性,INT显色检测LDH的释放,DCFH-DA荧光标记探针流式检测细胞活性氧代谢物(reactive oxygen species, ROS), PI(propidium iodide)流式检测亚二倍体峰(sub G1peak)。 2.采用Real Time PCR的方法检测MT2mRNA的表达。 3.采用重亚硫酸盐测序PCR的方法检测MT2、DAT和VMAT2基因启动子区域甲基化变化。 4.采用染色质免疫共沉淀的方法检测MT2、DAT和VMAT2基因启动子区域H3ac变化,最后进行real time定量PCR分析。结果1.HDACI对鱼藤酮和MPP+致多巴胺能神经细胞株损伤的影响。 (1)鱼藤酮和MPP+能显著降低多巴胺能神经细胞株活性。 (2) HDACI显著降低多巴胺能神经细胞株活性氧水平。 (3) HDACI显著降低鱼藤酮和MPP+诱导的多巴胺能神经细胞株凋亡 2. HDACI上调MT2mRNA表达。 3. HDACI降低MT2、DAT、VMAT2基因启动子区域甲基化率。 4. HDACI增加MT2、DAT、VMAT2基因启动子区域H3ac水平。结论HDACI(丁酸钠)对帕会森病具有保护作用,是通过表观遗传机制上调MT2和单胺转运体的表达实现。
[Abstract]:Background Parkinsonian disease (PD) is the second most common neurodegenerative disease characterized by a decrease in dopamine content in the substantia nigra and a loss of dopamine neurons in the substantia nigra. At present, the treatment of Parkinson's disease is mainly to maintain the balance of cholinergic and dopaminergic, alleviate motor symptoms, there is no cure. Oxidative stress and apoptosis are the most important theories in the pathogenesis of Parkinson's disease. Some studies have shown that the substantia nigra of PD patients is always in a state of high oxidative stress. Histone acetylation and deacetylation play an important role in Parkinson's disease and other neurodegenerative diseases. Histone deacetylase inhibitor (histone deacetylase inhibitor, HDACI has a protective effect on neurodegenerative diseases. Metallothionein 2 (MT2) is an oxidative stress resistance factor and histone deacetylase inhibitor can up-regulate the expression of MT2 gene. Dopamine transporter (dopamine transporter, DAT) and vesicular transporter (vesicular monoamine transporter, VMAT2) play a very important role in maintaining the balance of dopamine and other monoamines in the central nervous system. From 2% to 90% and related to gene expression, epigenetic regulation is an important mechanism of DAT and VMAT2 expression regulation. Therefore, does histone deacetylase inhibitor protect Parkinson's disease by upregulating the expression of MT2nDAT and VMAT2, and does its expression change through epigenetic mechanism? These need further study with us. Objective to investigate the protective effect of histone deacetylase inhibitor on Parkinson's disease cell model and its possible mechanism. Methods: dopaminergic nerve cell experimental group: control group (DMSO). The model group (0.5 渭 M rotenone 1 mm MPP 24 h) and sodium butyrate intervention group (1 mm M 2 mm MU 5 mm M + 48 h) 1. (reactive oxygen species, Ros and sub G1peak were detected by MTT colorimetry and sub G1peak, respectively. The expression of MT2 mRNA was detected by Real time PCR. The methylation changes of MT2DATand VMAT2 promoter region were detected by bisulfite sequencing PCR. 4. Chromatin immunoprecipitation was used to detect the H3ac changes in the promoter region of MT2DATand VMAT2 gene. Finally, real time quantitative PCR was used to detect the changes of H3ac in the promoter region of MT2DATand VMAT2 gene. Results 1. The effect of HDACI on the damage of dopaminergic nerve cell lines induced by rotenone and MPP. (1) rotenone and MPP significantly decreased the activity of dopaminergic nerve cell lines. (2) HDACI significantly decreased the activity of dopaminergic nerve cells. (3) HDACI significantly decreased the apoptosis of dopaminergic nerve cell lines induced by rotenone and MPP. HDACI upregulated MT2 mRNA expression. HDACI reduced the methylation rate of MT2 DATVMAT2 promoter region. 4. HDACI increased the level of H3ac in the promoter region of MT2 DATVMAT2 gene. Conclusion HDACI (sodium butyrate) has protective effect on Parsons disease, which is realized by upregulating the expression of MT2 and monoamine transporter by epigenetic mechanism.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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