组蛋白去乙酰化酶调控自噬对帕金森病保护作用的实验研究

发布时间：2018-05-21 02:18

本文选题：帕金森病 + 组蛋白去乙酰化酶　；参考：《华中科技大学》2016年博士论文

【摘要】：第一部分不同环境毒素诱导的PD细胞模型中HDAC亚型及PD相关重要分子变化目的研究PD环境毒素鱼藤酮或甲氰菊酯对拟多巴胺能SH-SY5Y细胞系HDAC不同亚型及PD相关重要蛋白表达及定位的影响。方法鱼藤酮或甲氰菊酯作用于SH-SY5Y细胞,采用Western blot或Real-time PCR方法了解环境毒素诱导的SH-SY5Y细胞模型中,HDAC1、HDAC4、HDAC6及PD标志性病理蛋白α-突触核蛋白(α-syn)的表达水平与处理时限或毒素浓度之间的关系；使用激光共聚焦显微镜观察鱼藤酮对HDAC不同亚型及α-syn定位及表达的影响。结果Western blot检测发现鱼藤酮处理可浓度依赖性及时间依赖性的诱发HDAC1、 HDAC4、HDAC6及α-syn蛋白表达增加,尤其寡聚体形式的α-syn增加明显。Real-timePCR检测表明,甲氰菊酯可浓度依赖性的诱发HDAC1、HDAC4、HDAC6和α-syn的mRNA水平升高,而多巴胺转运体(DAT)和囊泡单胺转运蛋白2(VMAT2)转录活性却随甲氰菊酯浓度增加呈现降低趋势。激光共聚焦显微镜观察发现,随着鱼藤酮处理时间延长,胞核定位的HDAC1荧光强度逐渐增强,并出现胞核内异常聚集。正常细胞中HDAC4均匀弥散分布于胞浆内,胞核内也有少量分布,鱼藤酮干预后HDAC4荧光强度明显增强,并出现胞浆内异常聚集以及胞核转位增加,且HDAC4与α-syn共定位明显。HDAC6与α-syn在正常细胞中弥散分布于细胞浆中,1μM鱼藤酮处理48小时后,HDAC6与α-syn荧光强度明显增强,并出现胞浆内异常聚集,HDAC6与α-syn蛋白共定位表达明显。结论环境毒素鱼藤酮或甲氰菊酯可诱导SH-SY5Y细胞中HDAC1、HDAC4、HDAC6及PD相关重要分子的表达及定位发生改变。第二部分不同环境毒素诱导的PD小鼠模型中HDAC亚型及PD相关重要分子变化目的研究PD环境毒素鱼藤酮或甲氰菊酯对C57BL6/J小鼠黑质区多巴胺能神经元HDAC不同亚型及PD相关重要蛋白表达及定位的影响。方法将老年C57BL6/J小鼠随机分为正常对照组和鱼藤酮造模组,持续8周予以鱼藤酮灌胃给药(30mg/kg/d),建立稳定可靠的PD小鼠模型,并采用激光共聚焦荧光显微镜观察中脑黑质致密区HDAC1、HDAC4、HDAC6以及a-syn、泛素的表达和定位变化。给予成年C57BL6/J小鼠腹腔注射甲氰菊酯,使用Real-time PCR方法检测小鼠中脑黑质区和纹状体区HDAC1、HDAC4、HDAC6和PD相关重要分子的表达变化与毒素浓度或暴露时间之间的关系。结果激光共聚焦荧光显微镜检测显示,长期慢性鱼藤酮干预后,小鼠黑质致密区多巴胺能神经元胞核内HDAC1表达较对照组明显增多,HDAC4表达也明显增加,部分细胞胞浆内出现HDAC4染色阳性的颗粒样聚集体。正常老年小鼠中HDAC6弥散分布于黑质多巴胺能神经元胞浆和神经突起中,慢性鱼藤酮刺激后神经元内HDAC6表达明显增多,神经突起内尤其明显,部分神经元胞浆内蛋白聚集增加。我们还发现,鱼藤酮干预后多巴胺能神经元胞浆和胞核中a-syn和泛素表达显著增多,部分细胞内出现a-syn或泛素染色阳性的粗颗粒样聚集。随着甲氰菊酯浓度升高或暴露时间延长,中脑黑质区a-syn转录活性呈增加趋势,而HDAC1、HDAC4、HDAC6以及DAT、 VMAT2的转录活性均呈下降趋势。此外,纹状体区DAT、VMAT2的转录活性也随甲氰菊酯处理时间延长逐渐降低。结论环境毒素鱼藤酮或甲氰菊酯干预可诱导小鼠多巴胺能神经元中HDAC1、 HDAC4、HDAC6及PD相关重要分子的表达及定位发生改变。第三部分HDAC抑制剂对鱼藤酮诱导的PD细胞模型的保护作用及相关机制目的探究不同HDAC抑制剂对鱼藤酮诱导的PD细胞模型的保护作用及可能的相关机制。方法采用Western blot方法,检测NaB或VPA对鱼藤酮诱导的SH-SY5Y及SK-N-SH细胞模型中α-syn寡聚体以及磷酸化水平的影响,同时检测自噬以及内质网应激相关指标的变化。并进一步采用不同浓度的NaB或VPA预处理细胞,了解这两种药物对鱼藤酮细胞模型的保护作用是否具有剂量依赖性。另外,分别使用3-MA或氯喹干扰自噬水平,Western blot检测上述指标变化,观察NaB或VPA对鱼藤酮诱导的PD细胞模型的影响。结果Western blot结果显示,NaB或VPA预处理能够明显减少鱼藤酮诱导的a-syn寡聚体和pSerl29 a-syn形成,同时上调Beclin 1表达,促进LC3-Ⅰ向LC3-Ⅱ转化,增强自噬活性,促进自噬流完成,并且减少鱼藤酮诱发的内质网应激对PD细胞模型发挥保护作用。这两种药物对鱼藤酮细胞模型的保护效应具有明显的剂量依赖性。我们还发现,NaB或VPA预处理上调LC3-Ⅱ表达这一效应被自噬抑制剂3-MA削弱。使用氯喹可抑制自噬流完成,LC3-Ⅱ表达明显增加,这一过程同样减弱了NaB或VPA增强自噬的作用。另外,无论是否加入3-MA或氯喹,NaB或VPA干预均能减少SK-N-SH细胞内pSer129 a-syn以及GRP78蛋白表达。结论NaB或VPA可通过增强自噬、促进自噬流完成以及减少内质网应激发挥对鱼藤酮细胞模型的保护作用。第四部分HDAC抑制剂对慢性鱼藤酮诱导的不同遗传背景PD小鼠模型的保护作用及相关机制目的探索HDAC抑制剂对慢性鱼藤酮诱导的不同遗传背景PD小鼠模型的保护作用及相关机制。方法将老年野生型C57BL6/J小鼠随机分为正常对照组、鱼藤酮造模组、VPA+鱼藤酮组以及NaB+鱼藤酮组,老年VMAT2+/-或DAT+/-杂合子小鼠随机分为溶剂对照组、鱼藤酮造模组以及NaB+鱼藤酮组,持续8周予以鱼藤酮灌胃以及VPA/NaB腹腔注射。通过Rotarod及爬杆实验评定小鼠运动及协调能力；高效液相色谱-电化学法检测小鼠纹状体DA神经递质含量；免疫组化、免疫荧光及Western blot检测小鼠黑质、纹状体区PD相关重要蛋白的表达及定位变化；透射电镜观察小鼠黑质和纹状体区神经元的超微结构。结果慢性鱼藤酮干预后不同遗传背景的小鼠较溶剂对照组爬杆时间明显延长,Rotarod潜伏时间缩短,其中VMAT2+/-、鼠运动功能减退较野生型小鼠更加显著,DAT+/-小鼠运动功能减退则不如野生型显著,而NaB或VPA预处理在3种基因型小鼠中均显著改善了鱼藤酮所致的运动功能损伤。高效液相色谱检测显示,NaB预处理均显著增加了3种基因型小鼠纹状体DA的含量。脑组织切片免疫组化及免疫荧光激光共聚焦检测显示,在野生型、VMAT2+/-和DAT+/-小鼠中,NaB或VPA预处理能部分逆转慢性鱼藤酮暴露所致的黑质致密部TH阳性神经元数目的减少,减轻黑质DA神经元和海马神经元内α-syn或泛素阳性的蛋白聚集,还能明显减少鱼藤酮引起的P62阳性颗粒样聚集物增多。Westem blot结果表明慢性鱼藤酮刺激后LC3-Ⅱ和P62蛋白水平增加,自噬流受阻,而NaB或VPA预处理通过增加LC3-Ⅱ表达,降低P62蛋白水平从而促进自噬流完成。透射电镜结果显示,慢性鱼藤酮干预后野生型及DAT+/-小鼠黑质区和纹状体区线粒体肿胀明显,部分线粒体嵴消失,甚至空泡化；NaB预处理可明显减轻鱼藤酮所致的线粒体损伤,并增加自噬液泡数量,尤其自噬溶酶体数目增加明显。结论HDAC抑制剂NaB通过增强自噬活性对慢性鱼藤酮诱导的不同遗传背景小鼠的黑质纹状体系统损伤具有明显的神经保护作用。
[Abstract]:The effects of PD environmental toxin rotenone or fenprothrin on the expression and localization of the different subtypes of HDAC and PD related important proteins in the pseudo dopaminergic SH-SY5Y cell line and the effect of PD on the expression and localization of PD related important proteins in the PD cell model of different environmental toxin induced PD cell models. To understand the relationship between the expression level of HDAC1, HDAC4, HDAC6 and PD ICP - alpha synuclein (alpha -syn) in the SH-SY5Y cell model induced by environmental toxins by Western blot or Real-time PCR, and the relationship between the time limit of treatment and the concentration of toxin; using laser confocal microscopy to observe the localization of rotenone to HDAC subtypes and alpha -syn Results Western blot detection found that rotenone treated the concentration dependent and time dependent induced HDAC1, HDAC4, HDAC6 and alpha -syn protein expression increased, especially in the oligomer form of alpha -syn, and the obvious.Real-timePCR detection showed that the concentration of pyrethrin could induce HDAC1, HDAC4, HDAC6 and alpha -syn. The transcriptional activity of the dopamine transporter (DAT) and the vesicle monoamine transporter 2 (VMAT2) decreased with the increase of the concentration of pyrethrin. The laser confocal microscope observed that the HDAC1 fluorescence intensity of the nucleus localization gradually increased with the prolonged treatment of rotenone, and the abnormal aggregation of the nucleus in the nucleus. HDAC4 in normal cells. The uniform dispersion was distributed in the cytoplasm, and there was a small amount of distribution in the nucleus. The intensity of HDAC4 fluorescence was obviously enhanced after rotenone, and the abnormal aggregation of the cytoplasm and the transposition of the nucleus appeared in the cytoplasm, and the HDAC4 and the alpha -syn were conformed with the obvious.HDAC6 and the alpha -syn in the normal cells and distributed in the cytoplasm. After 48 hours treatment of 1 UG, HDAC6 and alpha -sy were found. The fluorescence intensity of N was obviously enhanced and abnormal aggregation in the cytoplasm was found, and the expression of HDAC6 and alpha -syn protein was obvious. Conclusion the expression and localization of HDAC1, HDAC4, HDAC6 and PD related important molecules in SH-SY5Y cells can be induced by environmental toxin rotenone or fenpromethrin. HDAC in the PD mouse model induced by second different environmental toxins. Objective to study the effects of subtype and PD related important molecular changes in PD environmental toxin rotenone or fenprothrin on the expression and localization of HDAC different subtypes of dopaminergic neurons in the substantia nigra area of C57BL6/J mice and the expression and location of PD related important proteins. Methods the aged C57BL6/J mice were randomly divided into normal control group and To module, and continued to use rotenone for 8 weeks. A stable and reliable PD mouse model was established by intragastric administration (30mg/kg/d). The expression and localization of ubiquitin, HDAC1, HDAC4, HDAC6 and a-syn in the mesencephalic dense region were observed by laser confocal fluorescence microscopy. The intraperitoneal injection of fenpromethrin in adult C57BL6/J mice was given and Real-time PCR method was used to detect the black mass and striatum in the midbrain of mice. The relationship between the expression changes of HDAC1, HDAC4, HDAC6 and PD related important molecules with the concentration of toxin and the time of exposure. Results the laser confocal fluorescence microscopy showed that the prognosis of chronic rotenone showed that the HDAC1 table in the nucleus of dopaminergic neurons in the dense area of the murine substantia nigra was significantly increased, and the expression of HDAC4 increased obviously. In addition, in the cytoplasm of some cells, HDAC4 staining positive particle aggregates were found. In normal aged mice, HDAC6 dispersion was distributed in the cytoplasm and neurites of dopaminergic neurons in the substantia nigra. The expression of HDAC6 in neurons increased significantly in neurons after chronic rotenone stimulation, especially in the neurites, and the accumulation of protein in the cytoplasm of some neurons increased. We also found that after the intervention of rotenone, the expression of a-syn and ubiquitin in the cytoplasm and nucleus of dopaminergic neurons increased significantly. In some cells, there were a-syn or ubiquitin positive coarse particle like aggregation. With the increase of the concentration of methrin or the prolonged exposure time, the activity of a-syn transcriptional activity in the mesencephalic substantia nigra increased, while HDAC1, HDAC4, HDAC6 and D were increased. The transcriptional activity of AT and VMAT2 decreased. In addition, the transcriptional activity of DAT and VMAT2 in the striatum also gradually decreased with the prolongation of the time of methrin treatment. Conclusion the intervention of rotenone or pyrethrin can induce the expression and localization of HDAC1, HDAC4, HDAC6 and PD related important molecules in the dopaminergic neurons of mice. The three part of the protective effect of HDAC inhibitor on rotenone induced PD cell model and related mechanisms to explore the protective effect and possible mechanism of different HDAC inhibitors on rotenone induced PD cell model. Methods the Western blot method was used to detect the alpha -syn in the SH-SY5Y and SK-N-SH cell models induced by rotenone by NaB or VPA. The effects of oligomers and phosphorylation levels, and the changes in autophagy and endoplasmic reticulum stress related indicators, and the further use of different concentrations of NaB or VPA pretreated cells to understand whether the protective effects of these two drugs on rotenone cell model are dose-dependent. Furthermore, the use of 3-MA or chloroquine to interfere with autophagy, W Estern blot detected the changes of the above indexes and observed the effects of NaB or VPA on the PD cell model induced by rotenone. Results Western blot results showed that NaB or VPA pretreatment could significantly reduce the formation of a-syn oligomer and pSerl29 a-syn induced by rotenone, and up up the expression of 1. The autophagic flow was completed and the rotenone induced endoplasmic reticulum stress was protected against the PD cell model. The protective effects of these two drugs on the rotenone cell model were significantly dose-dependent. We also found that the effect of NaB or VPA preconditioning on the expression of LC3- II was weakened by the autophagic inhibitor 3-MA. Chloroquine was inhibited. The expression of autophagic flow was completed, the expression of LC3- II increased significantly. This process also weakened the role of NaB or VPA to enhance autophagy. In addition, the NaB or VPA intervention could reduce the pSer129 a-syn and GRP78 protein expression in SK-N-SH cells whether or not 3-MA or chloroquine. Conclusion NaB or VPA can enhance autophagy, promote autophagy completion and reduce endoplasmic reticulum. The protective effect of the net on rotenone cell model. Fourth the protective effect of HDAC inhibitor on the different genetic background PD mice induced by chronic rotenone and the related mechanisms to explore the protective effect and mechanism of HDAC inhibitors on different genetic background PD mice induced by chronic rotenone. The wild type C57BL6/J mice were randomly divided into normal control group, To module, VPA+ rotenone group and NaB+ rotenone group. The aged VMAT2+/- or DAT+/- heterozygote mice were randomly divided into the solvent control group, the To module and the NaB+ rotenone group, which lasted for 8 weeks with rotenone gavage and VPA/NaB intraperitoneal injection. Through Rotarod and climbing pole real. The activity and coordination ability of mice were evaluated and the content of DA neurotransmitter in mice striatum was detected by high performance liquid chromatography electrochemistry; immunohistochemistry, immunofluorescence and Western blot were used to detect murine substantia nigra, expression and localization of PD related proteins in striatum; ultrastructure of neurons in substantia nigra and striatum of mice was observed by transmission electron microscopy Results in mice with different genetic background, the climbing pole time of the mice with different genetic background was significantly longer than that of the solvent control group, and the latency of Rotarod was shortened, in which VMAT2+/-, the motor function decreased more significantly in mice than in the wild type mice, and the decrease of motor function in DAT+/- mice was not as significant as that of the wild type, while the NaB or VPA pretreatment was in 3 genotypic mice. The motor function damage caused by rotenone was significantly improved. High performance liquid chromatography (HPLC) showed that NaB preconditioning significantly increased the content of DA in the striatum of 3 genotypes. Immunohistochemistry of brain tissue and confocal immunofluorescence laser confocal microscopy showed that NaB or VPA pretreatment could be partially reversed in wild type, VMAT2+/- and DAT+/- mice. The number of TH positive neurons in the dense part of substantia nigra caused by chronic rotenone exposure decreased, alleviated the aggregation of alpha -syn or ubiquitin positive protein in DA neurons and hippocampal neurons, and significantly reduced the increase of P62 positive particles like aggregates caused by rotenone,.Westem blot results indicated that chronic rotenone stimulated LC3- II and P62 protein water. In addition, autophagic flow was hindered, while NaB or VPA preconditioning could promote autophagic flow by increasing LC3- II expression, reducing P62 protein level and promoting autophagic flow. The transmission electron microscopy showed that the mitochondria swelling in the substantia nigra and striatum of the wild type and DAT+/- mice was obvious, and the mitochondrial crista disappeared and even vacuolated, and NaB preconditioning could be used for the transmission electron microscope. The number of autophagic vacuoles increased significantly, especially the number of autophagic lysosomes increased obviously. Conclusion the HDAC inhibitor NaB has a clear neuroprotective effect on the nigrostriatal system damage in mice with different genetic background induced by chronic rotenone.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R742.5

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