组蛋白修饰对甲基苯丙胺诱发的行为敏化的影响

发布时间：2018-05-29 12:01

本文选题：甲基苯丙胺 + 行为敏化　；参考：《中国人民解放军军事医学科学院》2014年博士论文

【摘要】：药物成瘾是世界性难题，极大地损害人类的身心健康，影响社会稳定，增加医疗和经济负担。近年来，以甲基苯丙胺为代表的新型合成毒品滥用日趋严重，已成为重大的社会和公共卫生问题，研究其神经生物学机制以及寻找有效的诊断治疗方法迫在眉睫。药物成瘾是一种慢性复发性脑疾病，具有强迫用药和觅药的典型特点。一旦成瘾，终生难以摆脱，即使戒断较长时间后仍易于复吸。近年来的研究认为，药物成瘾是机体长期接触成瘾性药物后，中枢神经系统的神经可塑性发生异常改变，最终诱发成瘾行为。越来越多证据表明，表观遗传学是调控神经可塑性的一种重要方式，它不依赖于DNA序列改变而通过其他生化过程使机体内某些基因表达终生改变。这种表观遗传学修饰极其稳定，使其足以调控成瘾易感性和药物所诱发的代偿性适应。已有研究也证实，表观遗传学与药物成瘾密切相关，如急性或长期给予可卡因和其他成瘾性物质增加伏隔核（nucleus accumbens，NAc）内组蛋白H3、H4整体乙酰化水平；另外成瘾性药物也可改变一系列的表观遗传修饰调控酶。近年来，由于甲基苯丙胺滥用日趋严重，越来越多的科研人员开始研究其成瘾机制。本课题从组蛋白修饰角度—一种表观遗传学修饰研究甲基苯丙胺成瘾机制，以进一步加深对其机制的理解。一、甲基苯丙胺诱发的行为敏化模型反复给予成瘾性药物使得中枢神经系统对药物和药物相关刺激超敏，从而介导觅药和用药行为介导觅药和用药行为，表明敏化在药物复吸中发挥重要作用，并且二者具有共同的神经通路即前额叶皮层（prefrontal cortex，PFC）至中脑腹侧被盖区（ventral tegmental area，VTA）和NAc的投射，因此，本论文选定行为敏化模型研究甲基苯丙胺成瘾。行为敏化模型可分为形成、转化和表达三个阶段，本部分对形成和激发期（表达期）甲基苯丙胺给药剂量从0.25mg/kg-5.0mg/kg进行一系列的筛选，结合敏化后自发活动的变化，确定模型处理方案为连续7天给予5.0mg/kg甲基苯丙胺（s.c.，qd）后戒断7天，第15天给予1.0mg/kg甲基苯丙胺（s.c.）激发。甲基苯丙胺处理组（5.0mg/kg，s.c.，qd，7天）大鼠激发后水平活动距离明显高于生理盐水对照组大鼠（约30%），表明行为敏化模型建立成功。二、mRNA表达和组蛋白修饰的差异筛选本课题首次采用mRNA表达谱芯片和组蛋白修饰谱芯片研究甲基苯丙胺诱发的行为敏化大鼠PFC的mRNA差异表达及组蛋白修饰的改变，从转录和转录前水平分析该模型所诱发的变化。mRNA表达谱芯片筛选结果显示，共505个基因出现mRNA差异表达。具体的差异表达基因显示，在甲基苯丙胺处理后（急性给药后、转化期和表达期）mRNA表达高于盐水组，提示甲基苯丙胺活化多种基因转录；而甲基苯丙胺激发后mRNA高度表达也表明基因转录活化在敏化的表达中可能发挥了重要作用。GO分析将变化基因主要分为金属离子结合相关分子、调节转录分子、染色质重塑与DNA结合相关分子、细胞表面受体信号转导分子、质膜相关分子、参与神经系统发育分子、细胞凋亡相关分子和癌症相关分子等七类，这些分子有可能在药物成瘾中发挥重要作用。组蛋白修饰谱中差异修饰基因较多，主要变化位点为组蛋白乙酰化，2491个基因启动子位点H3乙酰化改变，5551个基因启动子位点H4乙酰化改变，而H3甲基化修饰几乎不改变，表明在甲基苯丙胺诱发的行为敏化模型中，主要是组蛋白乙酰化参与转录调控。 mRNA表达芯片中mRNA表达增加基因明显多于减少基因，而组蛋白修饰谱中H3、H4乙酰化修饰程度也在给与甲基苯丙胺后显著增加，提示组蛋白乙酰化修饰的增加介导基因转录的活化。综合分析mRNA表达和/或组蛋白修饰、基因功能、信号通路及基因在PFC内的基础表达量，选定Anp32a、Arrdc1、Atp5i、Avp、Bal2l1、Bdnf、Cadm3、Camk2n1、Cox6a1、Cox8a、E2f3、Egr1、Eml2、Exog、Galnt9、Hira、Limk1、Lnx2、Metrn、Ndst4、Nmur1、Oma1、Pou3f2、Shoc2、Stk32c、Stx2、Syt8、Tacr2、Trim17、Usp9x、Zfp36等共31个分子进行验证。三、转录上游分析组蛋白乙酰化修饰主要受两类酶调节：组蛋白乙酰转移酶（histone acetyltransferase，HAT）和组蛋白去乙酰化酶（histone deacetylase，HDAC）。在此模型中，大鼠PFC的CBP在激发后mRNA表达量下降至盐水对照的80%左右，HDAC1、2（属于I型Hdac）主要在甲基苯丙胺急性给药以及形成期慢性给药后下降，HDAC4、5（属于II型HDAC）则主要是在戒断（转化期）和激发后下降，提示不同类型的Hdac在此模型不同阶段发挥功能。HDAC活性则主要是在长期给药后降低，戒断和激发后显著增加。组蛋白修饰谱芯片提示，甲基苯丙胺处理后（急性给药后、转化期和表达期）总体组蛋白乙酰化明显增加，并且1.0mg/kg甲基苯丙胺激发后，组蛋白乙酰化修饰明显增加，与HDAC活性结果相反。上述结果的差异存在两种可能①本课题未研究的HAT和HDAC在此模型中发挥功能；②某一种或几种而非全部HDAC亚型参与此模型调控。这也为成瘾的进一步研究提供了线索和证据。四、候选分子验证及Pou3f2等基因在甲基苯丙胺诱发的行为敏化模型中转录和转录前的动态变化经过mRNA和组蛋白修饰两方面验证，选定Anp32a、Egr1、Eml2和Pou3f2四个分子作为候选分子，研究候选分子在甲基苯丙胺诱发的行为敏化模型各个阶段的动态变化。结果显示，Anp32a、Pou3f2组蛋白修饰与mRNA表达变化较一致，在整个敏化过程中，，急性及形成期给药使其组蛋白修饰和mRNA表达增加，戒断后恢复至正常水平，激发后显著增加。Egr1、Eml2组蛋白修饰与mRNA表达则不完全一致。Egr1在急性给药后及敏化形成期H4乙酰化有所增加，但mRNA表达量反而下降；7天戒断及激发后，Egr1组蛋白修饰和mRNA表达均无明显变化。Eml2在急性给药后H4组蛋白乙酰化增加，而mRNA显著降低；形成期给药后组蛋白修饰和mRNA表达均有一定程度的上升；戒断后，Eml2启动子位点H3、H4乙酰化修饰显著增加，但mRNA表达与盐水组无差异；而激发后，组蛋白修饰和mRNA表达均无明显变化。这些结果说明，组蛋白乙酰化修饰只是调控转录的一种方式，参与但并不一定决定基因转录能力。在上述候选分子中，在甲基苯丙胺成瘾的关键阶段，Pou3f呈现较明显的变化。文献表明，Pou3f2作为脑内特异表达的一个转录因子，与成瘾中研究较多的分子和通路（例如cAMP通路、Notch通路、MAPK通路等）密切相关，但是目前尚未研究Pou3f2本身与成瘾的关系。因此，本课题进一步研究Pou3f2与成瘾的关系。五、Pou3f2与甲基苯丙胺诱发行为敏化的相关性本部分成功建立了干涉Pou3f2的慢病毒。PFC亚区缘前皮层（prelimbiccortex，PL）注射慢病毒后建立甲基苯丙胺诱发的行为敏化模型，初步研究显示下调大鼠PL区Pou3f2mRNA的表达使甲基苯丙胺诱发的行为敏化大鼠激发后自发活动量降低约50%，提示Pou3f2可能是调控甲基苯丙胺诱发的行为敏化的重要分子，有可能在药物成瘾中发挥重要作用。综上所述，本课题研究发现，在甲基苯丙胺诱发的行为敏化模型中，大鼠PFC的基因组整体组蛋白乙酰化水平增加并介导下游基因的开放表达；而在转录上游，甲基苯丙胺通过调控CBP和HDAC的mRNA表达和活性而动态调控组蛋白乙酰化水平，这些结果提示PFC的组蛋白乙酰化修饰可能在甲基苯丙胺成瘾中发挥重要作用。另外，甲基苯丙胺诱导PFC内转录因子Pou3f2启动子区组蛋白乙酰化修饰及其转录水平的改变，可能是行为敏化形成的重要基础。
[Abstract]:Drug addiction is a worldwide problem , which greatly affects the physical and mental health of mankind , affects social stability and increases medical and economic burden . In recent years , the new synthetic drug abuse represented by methamphetamine has become a major social and public health problem . It is urgent to study the neurobiological mechanism and find effective diagnosis and treatment method .

Drug addiction is a kind of chronic recurrent brain disease . It has the typical characteristics of compulsive drug use and drug - seeking . In recent years , the neuroplasticity of the central nervous system has changed abnormally after long - term exposure to addictive drugs .
In addition , addictive drugs can alter a series of apparent genetic modification regulatory enzymes .

In recent years , because methamphetamine abuse is becoming more and more serious , more and more researchers have begun to study their addiction mechanism . This topic studies methamphetamine addiction mechanism from the perspective of histone modification - an epigenetics modification to further deepen the understanding of its mechanism .

1 . Methamphetamine - induced behavioral sensitization model

The behavioral sensitization model was divided into three stages : formation , transformation and expression . The behavioral sensitization model can be divided into three stages : formation , transformation and expression .

II . Differential screening of mRNA expression and histone modification

The mRNA differential expression of PFC and the change of histone modification induced by methamphetamine were studied by mRNA expression profiling and histone modification in the first time . mRNA differential expression was analyzed from the transcriptional and pre - transcriptional level .
GO analysis is mainly divided into metal ion binding - related molecules , regulatory transcription molecules , chromatin remodeling and DNA binding - related molecules , cell surface receptor signal transduction molecules , membrane - related molecules , participation in nervous system development molecules , apoptosis - related molecules and cancer - related molecules , etc . These molecules may play an important role in drug addiction .

There were many different modified genes in histone modification . The main site was histone acetylation , 2491 gene promoter sites H3 acetylation changed , 5551 gene promoter sites H4 acetylation changed , and the H3 methylation modification was hardly changed , indicating that in the behavioral sensitization model induced by methamphetamine , histone acetylation was mainly involved in the transcription regulation .

The expression of H3 and H4 in mRNA expression chip was significantly higher than that of reducing gene . The degree of acetylation of H3 and H4 in histone modification was also increased significantly after administration of methamphetamine . It was suggested that histone acetylation modification could mediate gene transcription .

III . Upstream Analysis of Transcription

The histone acetylation modification is mainly regulated by two types of enzymes : histone acetyl transferase ( HDAC ) and histone deacetylating enzyme ( HDAC ) . In this model , the mRNA expression level of PFC in rat was decreased to about 80 % of the saline control . HDAC1 , 2 ( belonged to type I Hdac ) decreased after acute administration of methamphetamine and chronic administration in the formal phase .
( 2 ) It also provides clues and evidence for the further study of addiction , which provides clues and evidence for further study of addiction . Four , candidate molecule verification and Pou3f2 gene are transcribed and transcribed in the behavioral sensitization model induced by methamphetamine .

The results showed that the expression of histone modifications and mRNA in Egr1 , Egr1 , Eml2 and Pou3f2 was more consistent with the changes of mRNA expression . Egr1 , Eml2 histone modifications and mRNA expression were not completely consistent .
After 7 days of withdrawal and excitation , there was no significant change in Egr1 group protein modification and mRNA expression . Eml2 increased histone acetylation after acute administration , while mRNA decreased significantly ;
There was a certain increase in histone modification and mRNA expression after drug administration .
After withdrawal , the acetylation of H3 and H4 increased significantly in Eml2 promoter , but there was no difference between mRNA expression and saline group .
There was no significant change in histone modification and mRNA expression after excitation . These results indicated that histone acetylation was only a way to regulate transcription , which involved , but not necessarily , the ability of gene transcription .

In the above - mentioned candidate molecule , Pou3f2 is a transcription factor specifically expressed in the brain and is closely related to more molecules and pathways ( e.g . , cAMP pathway , Notch pathway , MAPK pathway , etc . ) in addiction , but the relationship between Pou3f2 and addiction is not yet been studied . Therefore , the relationship between Pou3f2 and addiction is further studied .

In this part , the slow virus of interference Pou3f2 was successfully established . The behavioral sensitization model induced by methamphetamine was established after injection of lentivirus . The preliminary study showed that the expression of Pou3f2mRNA in rat PL was reduced by about 50 % , suggesting that Pou3f2 might be an important molecule to regulate the behavioral sensitization induced by methamphetamine . It was suggested that Pou3f2 might play an important role in drug addiction .

In conclusion , the study found that in the behavioral sensitization model induced by methamphetamine , the histone acetylation level of PFC in rat was increased and the open expression of downstream gene was mediated ;
At the upstream of transcription , methamphetamine regulates histone acetylation levels dynamically by regulating the expression and activity of mRNA and activity of CBP and HDAC . These results suggest that histone acetylation of PFC may play an important role in the addiction of methamphetamine . In addition , methamphetamine - induced modification of histone acetylation in the promoter region of the PFC internal transcription factor Pou3f2 and its transcription level may be an important basis for behavioral sensitization .
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R96

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