本文選題：甲基苯丙胺 + 行為敏化�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年博士論文

【摘要】：藥物成癮是世界性難題，極大地?fù)p害人類的身心健康，影響社會(huì)穩(wěn)定，增加醫(yī)療和經(jīng)濟(jì)負(fù)擔(dān)。近年來，以甲基苯丙胺為代表的新型合成毒品濫用日趨嚴(yán)重，已成為重大的社會(huì)和公共衛(wèi)生問題，研究其神經(jīng)生物學(xué)機(jī)制以及尋找有效的診斷治療方法迫在眉睫。 藥物成癮是一種慢性復(fù)發(fā)性腦疾病，具有強(qiáng)迫用藥和覓藥的典型特點(diǎn)。一旦成癮，終生難以擺脫，即使戒斷較長時(shí)間后仍易于復(fù)吸。近年來的研究認(rèn)為，藥物成癮是機(jī)體長期接觸成癮性藥物后，中樞神經(jīng)系統(tǒng)的神經(jīng)可塑性發(fā)生異常改變，最終誘發(fā)成癮行為。越來越多證據(jù)表明，表觀遺傳學(xué)是調(diào)控神經(jīng)可塑性的一種重要方式，它不依賴于DNA序列改變而通過其他生化過程使機(jī)體內(nèi)某些基因表達(dá)終生改變。這種表觀遺傳學(xué)修飾極其穩(wěn)定，使其足以調(diào)控成癮易感性和藥物所誘發(fā)的代償性適應(yīng)。已有研究也證實(shí)，表觀遺傳學(xué)與藥物成癮密切相關(guān)，如急性或長期給予可卡因和其他成癮性物質(zhì)增加伏隔核（nucleus accumbens，NAc）內(nèi)組蛋白H3、H4整體乙酰化水平；另外成癮性藥物也可改變一系列的表觀遺傳修飾調(diào)控酶。 近年來，由于甲基苯丙胺濫用日趨嚴(yán)重，越來越多的科研人員開始研究其成癮機(jī)制。本課題從組蛋白修飾角度—一種表觀遺傳學(xué)修飾研究甲基苯丙胺成癮機(jī)制，以進(jìn)一步加深對(duì)其機(jī)制的理解。 一、甲基苯丙胺誘發(fā)的行為敏化模型 反復(fù)給予成癮性藥物使得中樞神經(jīng)系統(tǒng)對(duì)藥物和藥物相關(guān)刺激超敏，從而介導(dǎo)覓藥和用藥行為介導(dǎo)覓藥和用藥行為，表明敏化在藥物復(fù)吸中發(fā)揮重要作用，并且二者具有共同的神經(jīng)通路即前額葉皮層（prefrontal cortex，PFC）至中腦腹側(cè)被蓋區(qū)（ventral tegmental area，VTA）和NAc的投射，因此，本論文選定行為敏化模型研究甲基苯丙胺成癮。行為敏化模型可分為形成、轉(zhuǎn)化和表達(dá)三個(gè)階段，本部分對(duì)形成和激發(fā)期（表達(dá)期）甲基苯丙胺給藥劑量從0.25mg/kg-5.0mg/kg進(jìn)行一系列的篩選，結(jié)合敏化后自發(fā)活動(dòng)的變化，確定模型處理方案為連續(xù)7天給予5.0mg/kg甲基苯丙胺（s.c.，qd）后戒斷7天，第15天給予1.0mg/kg甲基苯丙胺（s.c.）激發(fā)。甲基苯丙胺處理組（5.0mg/kg，s.c.，qd，7天）大鼠激發(fā)后水平活動(dòng)距離明顯高于生理鹽水對(duì)照組大鼠（約30%），表明行為敏化模型建立成功。 二、mRNA表達(dá)和組蛋白修飾的差異篩選 本課題首次采用mRNA表達(dá)譜芯片和組蛋白修飾譜芯片研究甲基苯丙胺誘發(fā)的行為敏化大鼠PFC的mRNA差異表達(dá)及組蛋白修飾的改變，從轉(zhuǎn)錄和轉(zhuǎn)錄前水平分析該模型所誘發(fā)的變化。mRNA表達(dá)譜芯片篩選結(jié)果顯示，共505個(gè)基因出現(xiàn)mRNA差異表達(dá)。具體的差異表達(dá)基因顯示，在甲基苯丙胺處理后（急性給藥后、轉(zhuǎn)化期和表達(dá)期）mRNA表達(dá)高于鹽水組，提示甲基苯丙胺活化多種基因轉(zhuǎn)錄；而甲基苯丙胺激發(fā)后mRNA高度表達(dá)也表明基因轉(zhuǎn)錄活化在敏化的表達(dá)中可能發(fā)揮了重要作用。GO分析將變化基因主要分為金屬離子結(jié)合相關(guān)分子、調(diào)節(jié)轉(zhuǎn)錄分子、染色質(zhì)重塑與DNA結(jié)合相關(guān)分子、細(xì)胞表面受體信號(hào)轉(zhuǎn)導(dǎo)分子、質(zhì)膜相關(guān)分子、參與神經(jīng)系統(tǒng)發(fā)育分子、細(xì)胞凋亡相關(guān)分子和癌癥相關(guān)分子等七類，這些分子有可能在藥物成癮中發(fā)揮重要作用。 組蛋白修飾譜中差異修飾基因較多，主要變化位點(diǎn)為組蛋白乙酰化，2491個(gè)基因啟動(dòng)子位點(diǎn)H3乙�；淖�，5551個(gè)基因啟動(dòng)子位點(diǎn)H4乙�；淖�，而H3甲基化修飾幾乎不改變，表明在甲基苯丙胺誘發(fā)的行為敏化模型中，主要是組蛋白乙�；瘏⑴c轉(zhuǎn)錄調(diào)控。 mRNA表達(dá)芯片中mRNA表達(dá)增加基因明顯多于減少基因，而組蛋白修飾譜中H3、H4乙�；揎棾潭纫苍诮o與甲基苯丙胺后顯著增加，提示組蛋白乙�；揎椀脑黾咏閷�(dǎo)基因轉(zhuǎn)錄的活化。綜合分析mRNA表達(dá)和/或組蛋白修飾、基因功能、信號(hào)通路及基因在PFC內(nèi)的基礎(chǔ)表達(dá)量，選定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個(gè)分子進(jìn)行驗(yàn)證。 三、轉(zhuǎn)錄上游分析 組蛋白乙�；揎椫饕軆深惷刚{(diào)節(jié)：組蛋白乙酰轉(zhuǎn)移酶（histone acetyltransferase，HAT）和組蛋白去乙�；福╤istone deacetylase，HDAC）。在此模型中，大鼠PFC的CBP在激發(fā)后mRNA表達(dá)量下降至鹽水對(duì)照的80%左右，HDAC1、2（屬于I型Hdac）主要在甲基苯丙胺急性給藥以及形成期慢性給藥后下降，HDAC4、5（屬于II型HDAC）則主要是在戒斷（轉(zhuǎn)化期）和激發(fā)后下降，提示不同類型的Hdac在此模型不同階段發(fā)揮功能。HDAC活性則主要是在長期給藥后降低，戒斷和激發(fā)后顯著增加。組蛋白修飾譜芯片提示，甲基苯丙胺處理后（急性給藥后、轉(zhuǎn)化期和表達(dá)期）總體組蛋白乙�；黠@增加，并且1.0mg/kg甲基苯丙胺激發(fā)后，組蛋白乙�；揎椕黠@增加，與HDAC活性結(jié)果相反。上述結(jié)果的差異存在兩種可能①本課題未研究的HAT和HDAC在此模型中發(fā)揮功能；②某一種或幾種而非全部HDAC亞型參與此模型調(diào)控。這也為成癮的進(jìn)一步研究提供了線索和證據(jù)。四、候選分子驗(yàn)證及Pou3f2等基因在甲基苯丙胺誘發(fā)的行為敏化模型中轉(zhuǎn)錄和轉(zhuǎn)錄前的動(dòng)態(tài)變化 經(jīng)過mRNA和組蛋白修飾兩方面驗(yàn)證，選定Anp32a、Egr1、Eml2和Pou3f2四個(gè)分子作為候選分子，研究候選分子在甲基苯丙胺誘發(fā)的行為敏化模型各個(gè)階段的動(dòng)態(tài)變化。結(jié)果顯示，Anp32a、Pou3f2組蛋白修飾與mRNA表達(dá)變化較一致，在整個(gè)敏化過程中，，急性及形成期給藥使其組蛋白修飾和mRNA表達(dá)增加，戒斷后恢復(fù)至正常水平，激發(fā)后顯著增加。Egr1、Eml2組蛋白修飾與mRNA表達(dá)則不完全一致。Egr1在急性給藥后及敏化形成期H4乙�；兴黾�，但mRNA表達(dá)量反而下降；7天戒斷及激發(fā)后，Egr1組蛋白修飾和mRNA表達(dá)均無明顯變化。Eml2在急性給藥后H4組蛋白乙�；黾�，而mRNA顯著降低；形成期給藥后組蛋白修飾和mRNA表達(dá)均有一定程度的上升；戒斷后，Eml2啟動(dòng)子位點(diǎn)H3、H4乙�；揎楋@著增加，但mRNA表達(dá)與鹽水組無差異；而激發(fā)后，組蛋白修飾和mRNA表達(dá)均無明顯變化。這些結(jié)果說明，組蛋白乙�；揎椫皇钦{(diào)控轉(zhuǎn)錄的一種方式，參與但并不一定決定基因轉(zhuǎn)錄能力。 在上述候選分子中，在甲基苯丙胺成癮的關(guān)鍵階段，Pou3f呈現(xiàn)較明顯的變化。文獻(xiàn)表明，Pou3f2作為腦內(nèi)特異表達(dá)的一個(gè)轉(zhuǎn)錄因子，與成癮中研究較多的分子和通路（例如cAMP通路、Notch通路、MAPK通路等）密切相關(guān)，但是目前尚未研究Pou3f2本身與成癮的關(guān)系。因此，本課題進(jìn)一步研究Pou3f2與成癮的關(guān)系。五、Pou3f2與甲基苯丙胺誘發(fā)行為敏化的相關(guān)性 本部分成功建立了干涉Pou3f2的慢病毒。PFC亞區(qū)緣前皮層（prelimbiccortex，PL）注射慢病毒后建立甲基苯丙胺誘發(fā)的行為敏化模型，初步研究顯示下調(diào)大鼠PL區(qū)Pou3f2mRNA的表達(dá)使甲基苯丙胺誘發(fā)的行為敏化大鼠激發(fā)后自發(fā)活動(dòng)量降低約50%，提示Pou3f2可能是調(diào)控甲基苯丙胺誘發(fā)的行為敏化的重要分子，有可能在藥物成癮中發(fā)揮重要作用。 綜上所述，本課題研究發(fā)現(xiàn)，在甲基苯丙胺誘發(fā)的行為敏化模型中，大鼠PFC的基因組整體組蛋白乙�；皆黾硬⒔閷�(dǎo)下游基因的開放表達(dá)；而在轉(zhuǎn)錄上游，甲基苯丙胺通過調(diào)控CBP和HDAC的mRNA表達(dá)和活性而動(dòng)態(tài)調(diào)控組蛋白乙�；�，這些結(jié)果提示PFC的組蛋白乙�；揎椏赡茉诩谆奖烦砂a中發(fā)揮重要作用。另外，甲基苯丙胺誘導(dǎo)PFC內(nèi)轉(zhuǎn)錄因子Pou3f2啟動(dòng)子區(qū)組蛋白乙�；揎椉捌滢D(zhuǎn)錄水平的改變，可能是行為敏化形成的重要基礎(chǔ)。
[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 .
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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