EFFECTS OF ALCOHOL RECOGNITION OF NEURAL STEM CELLS

神经干细胞酒精识别的影响

• 批准号: 7038372
• 负责人: WOJCIECH MICHAEL ZAWADA
• 金额: $ 11.46万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7038372
• 关键词: CD95 molecule; alcoholism /alcohol abuse; cell differentiation; cell proliferation; ethanol; free radical oxygen; immunocytochemistry; laboratory mouse; nerve stem cell; neuroimmunomodulation; neuroprotectants; neurotoxicology; neurotoxins; oxidative stress; tissue /cell culture; transfection; western blottings

DESCRIPTION (provided by applicant): Recent evidence shows that neural stem cell (NSC) proliferation and function are affected by alcohol. However, the mechanism of these effects is unknown. Alcohol induces oxidative stress, which will be considered as a potential mechanism for the alcohol's actions on NSCs in this application. Our recent work has shown that oxidative stress causes normally non-immunogenic tissues to express costimulatory molecules capable of activating T cells. The novelty of the present application is derived from the observation that alcohol affects the immunogenicity of neural stem cells. The expression and function of the immunologically important molecules on the surface of NSCs are poorly understood. We have begun addressing these questions and found that immunomodulatory molecules such as CD95 (Fas receptor) and the co-stimulatory molecule B7.1 are present on the surface of NSCs of mouse derivation and are upregulated by oxygen radicals. The key hypothesis behind this application is that alcohol's neurotoxic effects on neural progenitors are mediated by the recognition and response of the immune system to stress-induced changes in NSCs. The specific aims of this proposal are: (1) Determine whether alcohol kills NSCs and how it affects the expression of immune molecules in NSCs. We will initially examine whether alcohol exposure kills cultured mouse C17.2 NSCs and mouse endogenous NSCs in vivo using well-established assays for cell death and apoptosis. We will then use the C17.2 NSC line to systematically examine the expression of immune recognition molecules in NSCs and determine the effects of alcohol on their expression. The emphasis will be placed on the examination of the expression levels of a death receptor (CD95), primary immune recognition molecules such as major histocompatibility complexes (MHCs) and co-stimulatory molecules such as B7 and CD40. The cell surface molecules will be quantitated using flow cytometry. (2) Examine whether protection from reactive oxygen intermediates promotes NSCs' survival and differentiation following alcohol exposure. In this aim, we will stably overexpress in a regulatable fashion a mouse mitochondrial uncoupling protein 2 (UCP-2) in C17.2 cells to generate C17.UCP-2 cell line. UCP-2 decreases reactive oxygen species inside mitochondria. We hypothesize that expression of UCP-2 will reduce those actions of ethanol that are mediated by oxidative stress. (3) Determine whether transplanted C17.UCP-2 cells are more resistant to alcohol than NSCs expressing only natural levels of UCP-2. To extend the in vitro findings from aim 2, we will examine the effects of alcohol in vivo by transplanting cell lines generated in aim 2 into brains of wild type mice and mice deficient in UCP-2. We will systematically explore the changes in the immune molecules on the surface of transplanted NSCs in animals maintained on ethanol diet. Following transplantation, grafted NSC survival and UCP-2 expression in the grafts will be assayed and compared to these measures in endogenous NSCs. Proposed studies will systematically evaluate the effects of alcohol on the dynamics of immune molecules' expression on NSCs and their function. Such findings will stimulate further research bridging fields of alcohol, stem cells and immunology.

描述(由申请人提供)： 最近的证据表明，酒精对神经干细胞(NSC)的增殖和功能有影响。然而，这些效应的机制尚不清楚。酒精诱导氧化应激，这将被认为是酒精对神经干细胞作用的一个潜在机制。我们最近的工作表明，氧化应激通常会导致非免疫原性组织表达能够激活T细胞的共刺激分子。本应用的新颖性来自于观察到酒精影响神经干细胞的免疫原性。神经干细胞表面免疫重要分子的表达和功能目前还知之甚少。我们已经开始解决这些问题，并发现免疫调节分子，如CD95(Fas受体)和共刺激分子B7.1存在于小鼠神经干细胞的表面，并被氧自由基上调。这一应用背后的关键假设是，酒精对神经前体细胞的神经毒性效应是通过免疫系统对应激诱导的神经干细胞变化的识别和反应而介导的。这项提案的具体目的是：(1)确定酒精是否会杀死神经干细胞，以及它如何影响神经干细胞中免疫分子的表达。我们将使用成熟的细胞死亡和凋亡检测方法，初步检测酒精暴露是否会在体内杀死培养的小鼠C17.2神经干细胞和小鼠内源性神经干细胞。然后，我们将使用C17.2神经干细胞系系统地检测神经干细胞中免疫识别分子的表达，并确定酒精对其表达的影响。重点将放在对死亡受体(CD95)、主要组织相容性复合体(MHC)等主要免疫识别分子以及B7和CD40等共刺激分子的表达水平的检测上。细胞表面分子将用流式细胞仪进行定量。(2)研究活性氧中间体对酒精暴露后神经干细胞的存活和分化是否有促进作用。为此，我们将以可调控的方式在C17.2细胞中稳定过表达小鼠线粒体解偶联蛋白2(UCP-2)，以产生C17.UCP-2细胞系。UCP-2减少线粒体内的活性氧物种。我们假设UCP-2的表达将减少由氧化应激介导的乙醇的作用。(3)确定移植的C17UCP-2细胞是否比仅表达自然水平的UCP-2的神经干细胞对酒精更具抵抗力。为了扩展Aim 2的体外发现，我们将通过将Aim 2产生的细胞系移植到野生型小鼠和UCP-2缺陷小鼠的大脑中来检查酒精在体内的影响。我们将系统地探讨酒精饮食动物移植神经干细胞表面免疫分子的变化。移植后，移植的神经干细胞存活和UCP-2在移植物中的表达将被检测，并在内源性神经干细胞中与这些措施进行比较。拟议的研究将系统地评估酒精对神经干细胞上免疫分子表达的动态及其功能的影响。这些发现将刺激酒精、干细胞和免疫学领域的进一步研究。