THE FUTURE IS NOW:STEM CELLS AND ALCOHOL

未来就在眼前：干细胞和酒精

• 批准号: 6449682
• 负责人: WOJCIECH MICHAEL ZAWADA
• 金额: $ 24.76万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-27 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6449682
• 关键词: alcoholic beverage consumption; amygdala; behavior test; behavioral /social science research tag; cooperative study; corpus striatum; dopamine; dopamine transporter; gene expression; glia; laboratory mouse; neurons; nucleus accumbens; pluripotent stem cells; prefrontal lobe /cortex; stem cell transplantation; substance abuse related behavior; tissue /cell culture; transfection

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.

描述（由申请人提供）： 大脑多巴胺被认为可以调节饮酒量。我们假设 细胞外多巴胺 (DA) 水平的降低会减少乙醇 摄入量。该提案的主要目标是检查是否可以移植 经修饰可过度表达人类 DA 转运蛋白的神经干细胞 (NSC) (hDAT) 进入特定的大脑部位可以降低细胞外 DA 水平并改变 乙醇的行为动作。 NSC 是存在于 发育中和成人的大脑。 NSC 有能力分化成所有 已知的神经类型细胞包括神经元、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它们成为遗传研究的理想候选者 改造并移植到中枢神经系统（CNS）。这 该提案将探索一种利用干细胞移植的新策略 调节酒精的影响和摄入量。本提案的具体目标 是： (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。因为细胞系表达高 不存在适合神经移植的 hDAT 水平，我们建议 生成表达 hDAT 的高神经干细胞系 (C17.hDAT) 移植。我们将使用小鼠 v-myc 永生化神经干细胞 (C17.2)从Evan Snyder博士获得作为细胞系开发平台。 (2) 检查C17.hDAT干细胞来源的hDAT表达和功能 神经元和神经胶质细胞。我们建议检查 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细胞。该目标还将审查 调节 Tet-On 系统驱动的 hDAT 表达和功能的有效性 与多西环素。 (3) 判断是否移植C17.hDAT干细胞 进入小鼠大脑可以减少体内细胞外多巴胺并改变乙醇浓度 行动。我们首先将目标1中开发的C17.hDAT细胞移植到 Marc Caron 博士提供的野生型 (wt) 和 DAT 敲除小鼠的大脑 并进入高酒精 (C57BL/6) 和低酒精 (DBA) 野生型小鼠的大脑 偏爱。移植的目标区域将包括伏隔核、 前额皮质、杏仁核和背侧纹状体。嫁接至 DAT 敲除 小鼠应该扭转减少的行为和生化后果 在 DAT 级别。移植细胞的存活和转基因 hDAT 的功能将 被检查。行为测试将检查乙醇诱导的运动 活动，小鼠将被出口到联盟的其他地点 测试酒精偏好。这些研究将产生表达 hDAT 的干细胞 细胞移植到中枢神经系统并检查干细胞疗法是否可以 减少酒精的作用。这些发现将为以下问题提供第一个证据： 使用干细胞治疗药物依赖。