THE FUTURE IS NOW:STEM CELLS AND ALCOHOL

描述（由申请人提供）： 大脑多巴胺被认为调节酒精消耗。我们假设 细胞外多巴胺（DA）水平的降低会减少乙醇 摄入这项建议的主要目的是检查是否移植 神经干细胞（NSC）被修饰以过表达人DA转运蛋白 （hDAT）进入特定的大脑部位可以降低细胞外DA水平， 乙醇？的行为。神经干细胞是存在于神经干细胞中的多能细胞。 发育中的和成年的大脑。神经干细胞有能力分化成所有 已知的神经型细胞包括神经元、星形胶质细胞和少突胶质细胞。 神经干细胞前所未有的可塑性使其成为遗传学研究的理想候选者。 修饰和移植到中枢神经系统（CNS）中。这 该提案将探索一种新的策略，使用干细胞移植， 缓和酒精的影响和摄入量。本提案的具体目标 （1）表达人DAT（hDAT）的神经干细胞系的产生 在诱导型启动子的控制下。因为高表达的细胞系 水平的hDAT适合神经移植不存在，我们建议 以产生hDAT表达高神经干细胞系（C17.hDAT）用于细胞培养， 移植我们将使用小鼠v-myc永生化神经干细胞 （C17.2）从Evan Snyder博士处获得，作为细胞系开发平台。 (2)检测C17.hDAT干细胞源性细胞中hDAT的表达和功能 神经元和神经胶质。我们建议检测hDAT在干细胞中的表达和功能， 细胞分化成神经元或神经胶质。这一目标还将审查 调节Tet-On系统驱动hDAT表达和功能的有效性 强力霉素(3)确定是否移植C17.hDAT干细胞 能减少体内细胞外多巴胺和改变乙醇？S 行动我们将首先将在aim 1中开发的C17.hDAT细胞移植到 野生型（wt）和DAT敲除小鼠的大脑，由Marc Caron博士提供 并进入具有高（C57 BL/6）和低（DBA）酒精的wt小鼠的脑中 偏好移植的目标区域包括髓核， 前额皮质杏仁核和背侧纹状体移植入DAT敲除 小鼠应该扭转减少的行为和生化后果， 在DAT水平。移植细胞的存活和转基因hDAT的功能将 接受检查。行为测试将检查乙醇诱导的运动 活动和小鼠将被出口到其他网站的财团， 酒精偏好测试这些研究将产生hDAT表达干细胞， 研究干细胞治疗是否能 减少酒精？的行动。这些发现将提供第一个证据， 干细胞在药物依赖治疗中的应用。

DESCRIPTION (provided by applicant): Brain dopamine is postulated to modulate alcohol consumption. We hypothesize that reduction in extracellular dopamine (DA) levels would reduce ethanol intake. The main goal of this proposal is to examine if transplantation of neural stem cells (NSCs) modified to overexpress the human DA transporter (hDAT) into specific brain sites can reduce extracellular DA levels and alter ethanol?s behavioral actions. NSCs are pluripotent cells that exist in the developing and adult brain. NSCs have a capacity to differentiate into all known neural type cells including neurons, astrocytes and oligodendrocytes. Unprecedented plasticity of NSCs makes them ideal candidates for genetic modification and transplantation into the central nervous system (CNS). This proposal will explore a novel strategy using stem cell transplantation for moderating alcohol effects and intake. The specific aims of this proposal are: (1) Generation of neural stem cell lines expressing human DAT (hDAT) under control of an inducible promoter. Because cell lines expressing high levels of hDAT suitable for neural transplantation do not exist, we propose to generate a hDAT-expressing high neural stem cell line (C17.hDAT) for cell transplantation. We will use mouse v-myc immortalized neural stem cells (C17.2) obtained from Dr. Evan Snyder as the cell line development platform. (2) Examine the hDAT expression and function in C17.hDAT stem cell-derived neurons and glia. We propose to examine hDAT expression and function in stem cells differentiated into neurons or glia. This aim will also examine the effectiveness of regulating Tet-On system-driven hDAT expression and function with doxycycline. (3) Determine if transplantation of C17.hDAT stem cells into mouse brain can reduce extracellular dopamine in vivo and alter ethanol?s actions. We will first transplant C17.hDAT cells developed in the aim 1 into the brains of wildtype (wt) and DAT knockout mice provided by Dr. Marc Caron and into the brains of wt mice with high (C57BL/6) and low (DBA) alcohol preference. Target areas for grafting will include nucleus accumbens, prefrontal cortex, amygdala and dorsal striatum. Grafting into DAT knockout mice should reverse the behavioral and biochemical consequences of reduction in DAT levels. Survival of grafted cells and function of transgenic hDAT will be examined. Behavioral testing will examine ethanol-induced locomotor activity and the mice will be exported to other sites in the consortium to test for alcohol preference. These studies will generate hDAT-expressing stem cells for transplantation in the CNS and examine whether stem cell therapy can reduce alcohol?s actions. Such findings will provide the first evidence for use of stem cells in the treatment of drug dependence.