腹外侧眶皮层组蛋白H3K9乙酰化调控吗啡成瘾记忆的作用机制研究

Histone acetylation is an important way of histone modifications which has become a new research perspective of drug addiction memory. Our recent results suggested that the ventrolateral orbital cortex (VLO) as a crucial brain region which involved in the regulation of morphine addiction. The histone H3 lysine 9 acetylation (aceH3K9) in the VLO has been found to be participated in the modulation of morphine-induced behavioral sensitization, but the roles of aceH3K9 in the VLO on morphine addiction memory and its mechanism is still not clearly understood. The present study using a rat model of morphine-induced conditioned place preference (CPP) simulates the environmental cues memory. Obtain the correlative changes of synaptic plasticity in the VLO neurons by using the transmission electron microscope. Detect the expression of aceH3K9 by using RT-PCR, Western blot. Construct lentiviral over-expression and interference vectors which were microinjected into the VLO is to manipulate the expression of aceH3K9 during different stages of morphine addiction. Observe the correlative behavior changes, ultrastructural changes in VLO' synapses and expression of memory-related genes identify the regulatory effects of aceH3K9 on addiction memory. The agonist and antagonist which are microinjected into the VLO to activate or inhibit the dopamine receptor (DR), detecting the expression of DR-ERK pathway and aceH3K9 in the VLO. The present study will not only clarify the molecular mechanisms of aceH3K9 in the VLO underlying morphine addiction memory, but also provide valuable experimental evidence and theoretical basis for the development of new treatment target in morphine addiction.

组蛋白乙酰化是组蛋白重要的修饰方式，已成为研究药物成瘾记忆新的视角。我们前期发现，腹外侧眶皮层（VLO）是调控吗啡成瘾记忆的重要核团，VLO组蛋白H3K9乙酰化（aceH3K9）参与吗啡诱导的大鼠行为敏化的调控，但其确切的调控机制尚不清楚。本课题建立大鼠条件性位置偏爱（CPP）模型模拟吗啡成瘾记忆过程，观察成瘾不同阶段VLO内神经元突触超微结构变化及aceH3K9的mRNA、蛋白表达；利用脑立体定位术在VLO内注射慢病毒载体，过表达/干扰aceH3K9的表达，观察CPP不同阶段动物行为学、VLO内突触超微结构及记忆相关基因的变化；在VLO内注射与成瘾密切相关的多巴胺受体（DR）的激动剂/拮抗剂，检测DR-ERK通路、aceH3K9的表达及其在VLO内的共表达定位。本课题将阐明VLO组蛋白H3K9乙酰化调控吗啡成瘾记忆的作用机制，也将为寻找吗啡成瘾治疗的新靶点提供理论依据。

药物成瘾是一种慢性复发性脑病，主要表现为强迫性用药和对药物的持续性渴求。由于成瘾的发生机制十分复杂，成瘾相关的神经生物学机制仍不清楚，目前尚无特别有效的治疗药物。探讨成瘾的神经生物学机制，寻找新的干预靶点，对开发新的治疗成瘾的药物具有重要意义。本项目建立行为敏化和CPP成瘾模型，应用脑立体定位手术，在VLO内微量注射选择性SIRT1抑制剂EX527，构建SIRT1过表达/干扰腺相关病毒载体，在VLO内微量注射腺相关病毒，过表达/干扰SIRT1在VLO内的表达，记录行为学变化，检测SIRT1及相关分子蛋白的表达，记录行为学变化，检测组蛋白H3K9的重要调节因子SIRT1及相关分子的表达；提取成瘾模型大鼠VLO组织，进行转录组测序及miRNA测序，分析差异表达的基因，结合行为学指标，探讨组蛋白乙酰化在吗啡成瘾中的作用及可能机制。结果显示，VLO内微量注射EX527可显著降低吗啡诱导的行为敏化，可能的机制是通过抑制SIRT1及其下游分子p-ERK、BDNF等的表达，进而参与成瘾行为的调节；VLO内微量注射SIRT1过表达相关病毒，可抑制CPP的形成；通过对吗啡CPP成瘾大鼠VLO组织转录组测序结果的差异分析，发现成瘾组与对照组共有2027个基因在转录水平存在差异，其中989个基因转录水平上调，1038个基因转录水平下调；通过对吗啡成瘾大鼠VLO组织进行miRNA测序，发现8个miRNA在吗啡成瘾组与对照组之间存在差异表达，其中，4个miRNA表达上调的，4个miRNA表达下调。以上结果证实，SIRT1作为VLO内重要的成瘾调节因子，其调控成瘾行为可能是通过VLO内组蛋白H3K9乙酰化参与吗啡成瘾记忆过程。本研究将为脑的高级中枢参与药物成瘾提供新的重要证据，有望为开发新型治疗成瘾药物开拓新的思路，具有重要的科学价值和潜在的应用前景。