THE FUTURE IS NOW:STEM CELLS AND ALCOHOL

未来就在眼前：干细胞和酒精

• 批准号: 6533682
• 负责人: WOJCIECH MICHAEL ZAWADA
• 金额: $ 25.53万
• 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/6533682
• 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）： 假设脑多巴胺可以调节饮酒。我们假设 细胞外多巴胺（DA）水平的降低将降低乙醇 进气。该提案的主要目的是检查移植 神经干细胞（NSC）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水平并改变 乙醇的行为行动。 NSC是存在于 发展和成人大脑。 NSC有能力分为所有 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。 NSC的前所未有的可塑性使其成为遗传的理想候选者 修饰和移植到中枢神经系统（CNS）中。这 提案将使用干细胞移植来探索一种新的策略 调节酒精效应和摄入量。该提议的具体目的 为：（1）表达人DAT的神经干细胞系（HDAT）的产生 在诱导启动子的控制下。因为细胞系表达高 我们建议不存在适合神经移植的HDAT水平 为细胞生成表达HDAT的高神经干细胞系（C17.HDAT） 移植。我们将使用小鼠V-MYC永生的神经干细胞 （C17.2）从Evan Snyder博士作为细胞线开发平台获得。 （2）检查C17.HDAT干细胞衍生的HDAT表达和功能 神经元和神经胶质。我们建议检查茎中的HDAT表达和功能 细胞分化为神经元或神经胶质。这个目标还将检查 调节TET-ON系统驱动的HDAT表达和功能的有效性 与强力霉素。 （3）确定C17.HDAT干细胞的移植是否是否 进入小鼠大脑可以减少体内细胞外多巴胺，并改变乙醇？ 动作。我们将首先移植C17.HDAT细胞在AIM 1中开发 Marc Caron博士提供的Wildtype（WT）和DAT淘汰小鼠的大脑 并进入高（C57BL/6）和低（DBA）酒精的WT小鼠的大脑 偏爱。嫁接的目标区域将包括伏隔核， 前额叶皮层，杏仁核和背纹状体。嫁接DAT淘汰赛 小鼠应扭转还原的行为和生化后果 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引起的运动器 活动和小鼠将出口到财团的其他地点 测试酒精偏爱。这些研究将产生表达HDAT的茎 细胞进行中枢神经系统的移植，并检查干细胞疗法是否可以 减少酒精的作用。这样的发现将为 干细胞在药物依赖性治疗中的使用。