miR-212/sema4G调控中脑边缘多巴胺亚群神经元靶向投射与甲基苯丙胺致心理渴求关系的研究

Methylamphetamine (MA) is the most common drug with high potential for abuse which is difficult to withdraw due to psychol dependence. During development of mammalian animal,dopamine neurons of midbrain form different projections, among which mesolimbic DA projection is the neurobiological basis for MA dependence. Drug addiction shares the common molecules which regulate mesolimbic projection. However, little is known about the molecular mechanism of regulation of mesolimbic projection. In our prelimanary study, mesolimbic DA subtype neurons were isolated by retrograde tracing and fluorescence activated cell sorting, specific miRNAs and genes were screened with microarray analysis of whole microRNA and gene expression. As the specific miRNA and gene of mesolimbic projection, miR-212 and sema4G were selected to further analyze their functions. The current proposal aims to 1) veritify the expression of miR-212 and sema4G, and confirm their roles in mesolimbic projection with gain-of function and loss-of function; 2) analyze the changes of MA craving of electrporation mice with mesolimbic projection interfered; 3)produce animal model with MA, and the microinjection of antisense oligodeoxynucleotide of candidate genes will be performed to further analyze their functions in animal model. The current project will clarify the molecular mechanism of mesolimbic projection and potentially lead to a new therapeutic approach for the treatment drug addiction to methylamphetamine.

甲基苯丙胺（MA）是最常见的毒品之一，沾染后难以戒除，原因是不能消除心理渴求。已知中脑边缘（mesolimbic）多巴胺投射是药物成瘾的结构基础，MA等物质过度激活mesolimbic投射引发心理渴求。目前缺乏的是发现mesolimbic投射的特异调控分子并研究与MA成瘾的关系。我们在预实验中应用Retrograde-FACS技术提取了中脑边缘DA亚群神经元，通过基因及miRNA表达谱分析，筛出了miR-212和轴突导向因子sema4G，且发现MA上调miR-212，同时下调sema4G。本项目拟：1）通过基因转染、RNAi及胎脑体内电转，从体外及体内分析miR-212和sema4G对mesolimbic投射的调控作用；2）干扰mesolimbic投射后在体检测MA行为敏化及渴求的变化；3）建立MA模型鼠进行干预。本项目将揭示mesolimbic投射的调控机制，有望找出MA渴求的分子靶点。

甲基苯丙胺（MA）是最常见的毒品之一，沾染后难以戒除的主要原因是不能消除心理渴求。已知中脑边缘多巴胺投射（mesolimbic projection）是药物成瘾的结构基础，MA等药物异常激活mesolimbic投射的特异调控分子是引发成瘾的机制。目前缺乏的是mesolimbic投射特异调控分子的发现并揭示其与MA成瘾关系的研究。为揭示mesolimbic投射与成瘾的机制，本项目按照原设计方案开展了相关的实验研究，完成了计划的实验内容。首先，我们示踪并提取了中脑边缘多巴胺能亚群神经元，分析了MA对其电生理学特性的影响，并延展实验分析了D2受体对Ih电流的影响。然后，通过miRNA和基因表达谱分析，筛选了中脑边缘投射神经通路差异表达miRNAs和特异表达基因，经生物信息学分析并参考以往的研究，选定了功能分析的候选分子。接下来，本研究构建了针对miR-212/132的过表达病毒载体和其靶基因的干扰病毒载体，并体外转染神经元，分析了差异miRNA-212/132和靶基因对MA致神经元损伤及相关分子通路的影响。最后，我们建立了MA行为敏化动物模型，检测了差异miRNAs和基因的表达变化，并通过病毒载体体内注射，进行了模型鼠干预实验，并拓展实验内容，观察了药物的干预作用。综上，本项研究从解剖学层面上，为理解MA致药物成瘾的神经解剖学基础及相关的分子机制提供了实验依据。相关研究成果已发表英文SCI论文和国内核心期刊论文，参加全国及省级学术会议交流，部分交流为会议报告，培养了本领域研究生，取得了预期的研究成果。