THE FUTURE IS NOW:STEM CELLS AND ALCOHOL

描述(由申请人提供)： 大脑中的多巴胺被认为是调节酒精消费的物质。我们假设 细胞外多巴胺(DA)水平的降低将减少乙醇 入口处。这项建议的主要目标是检查是否可以移植 高表达人DA转运蛋白的神经干细胞(NSCs) (HDAT)进入特定的大脑部位可以降低细胞外DA水平并改变 酒精？S的行为举止。神经干细胞是一种多能细胞，存在于 发育中的和成年的大脑。神经干细胞具有分化为 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理想的遗传候选者 修饰和移植到中枢神经系统(CNS)。这 提案将探索一种使用干细胞移植的新策略 调节酒精的影响和摄入量。这项建议的具体目的 (1)表达人DAT(HDAT)神经干细胞系的建立 在可诱导启动子的控制下。因为高表达的细胞系 我们建议，不存在适合神经移植的HDAT水平 建立高表达HDAT的神经干细胞系(C17.hDAT) 移植。我们将使用小鼠v-myc永生化神经干细胞 (C17.2)，取自Evan Snyder博士，作为细胞系发展平台。 (2)检测HDAT在C17.hDAT干细胞中的表达和功能 神经元和神经胶质细胞。我们建议研究HDAT在茎中的表达和功能 细胞分化为神经元或神经胶质细胞。这一目标还将审查 调控Tet-on系统驱动的HDAT表达和功能的有效性 多西环素。(3)确定C17.hDAT干细胞是否移植 进入小鼠脑内能否减少体内细胞外多巴胺并改变乙醇？S 行为。我们将首先将在Aim 1中培育的C17.hDAT细胞移植到 Marc Caron博士提供的野生型(Wt)和DAT基因敲除小鼠的大脑 并进入高(C57BL/6)和低(DBA)酒精的WT小鼠的脑内 偏好。移植的目标区域将包括伏隔核， 前额叶皮质、杏仁核和背侧纹状体。嫁接到DAT基因敲除 小鼠应该逆转还原的行为和生化后果 以DAT级别表示。移植细胞的存活和转基因HDAT的功能 接受检查。行为测试将检查酒精诱导的运动 活动和MICE将被出口到财团的其他地点，以 酒精偏好测试。这些研究将产生HDAT表达茎 用于中枢神经系统移植的细胞，并检查干细胞疗法是否可以 戒酒？S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.