Generation of High Passage and Immortalized Human Microglia Cell Lines

描述（由申请人提供）：拟定研究的目标是生成并广泛表征高传代和永生化成人小胶质细胞系。高产量和良好的特点成人小胶质细胞培养的每一个案件将是必要的机制在体外研究。大量细胞的可用性也将允许对可以调节小胶质细胞功能的药物进行初步测试，并允许分离大量RNA和/或蛋白质用于转录组学或蛋白质组学研究。小胶质细胞在健康和患病的大脑中具有不同的功能。关于小胶质细胞生物学的大量研究都是基于体外研究，其中绝大多数使用的是从啮齿动物大脑中分离出来的细胞。然而，人类大脑的更高的解剖学和功能复杂性以及小胶质细胞反应和功能的物种差异使得必须使用人类小胶质细胞来确定所获得的结果适用于man. Further，在成人大脑中的小胶质细胞功能的研究，其中发生许多炎症和抗炎小胶质细胞反应，需要使用来自成人大脑的人类小胶质细胞。从胚胎人脑培养的小胶质细胞显示出相当大的增殖能力。然而，虽然存在从成年死后人脑中分离小胶质细胞的方法，但由于增殖水平低，它们仅允许使用从每个病例中分离和培养的有限量的小胶质细胞。在初步的尝试中，我们已经开发出一种方法，允许培养小胶质细胞从成年人死后的人脑相对较高的通道。有限的初步测试表明，人类小胶质细胞可以保持其表型在高传代培养。拟议的工作将试图产生更高的传代培养的小胶质细胞和广泛的表征小胶质细胞的表型和不同的刺激在不同的通道。我们还将产生和广泛的特点永生化的人小胶质细胞系使用一个完善的协议，利用人端粒酶逆转录酶。所提出的工作的具体目标将测试以下假设：目标1 -从不同年龄的正常成人大脑和患有神经退行性疾病的个体的大脑中培养的小胶质细胞将在体外增殖到高传代。目的2 -高传代人小胶质细胞将保持其表型，包括对各种刺激物的反应。目的3 -人小胶质细胞将成功永生化，永生化的人小胶质细胞将保持其表型和对各种刺激物的反应。高传代和永生化的人类小胶质细胞培养物的成功产生和表征将为科学界提供新的可靠工具，用于人类小胶质细胞在从儿童到老年的健康和疾病中的功能的体外机制研究，包括我们自己对阿尔茨海默病中小胶质细胞功能的研究。

DESCRIPTION (provided by applicant): The goal of the proposed research is to generate and extensively characterize high passage and immortalized adult human microglia cell lines. High yield and well characterized adult human microglia cultures from each case will be necessary for mechanistic in vitro studies. Availability of large numbers of cells would also allow initial testing of drugs that can regulate microglial functions and permit isolation of large amounts of RNA and/or protein for transcriptomic or proteomic studies. Microglia have diverse functions in the healthy and diseased brain. A great deal of what has been learned regarding microglia biology is based on in vitro studies the overwhelming majority of which used cells isolated from the rodent brain. However, higher anatomical and functional complexity of the human brain and species differences in microglia response and function make imperative the use of human microglia to ascertain that the results obtained are applicable to man. Furthermore, investigation of microglia functions in the adult brain, in which many inflammatory and anti- inflammatory microglia responses occur, requires use of human microglia from adult brains. Microglia cultured from embryonic human brain show substantial proliferative capacity. However, while methods for isolation of microglia from adult postmortem human brains exist, they allow only use of a limited quantity of microglia isolated and cultured from each case due to low levels of proliferation. In preliminary attempts, we have developed a method that allows culturing microglia from adult postmortem human brains to relatively high passage. Limited preliminary testing indicated that human microglia may maintain their phenotype in high passage cultures. The proposed work will attempt to generate microglia cultures of higher passage and to extensively characterize microglia phenotypes and response to different stimuli at different passages. We will also generate and extensively characterize immortalized human microglia cell lines using a well-established protocol that utilizes human telomerase reverse transcriptase. The specific aims of the proposed work will test the following hypotheses: Aim 1 - Microglia cultured from normal adult human brains of various ages and from brains of individuals with neurodegenerative disorders will proliferate to high passage in vitro. Aim 2 - High passage human microglia will maintain their phenotype, including response to various stimulants. Aim 3 - Human microglia will be successfully immortalized, and immortalized human microglia will maintain their phenotype and response to various stimulants. Successful generation and characterization of high passage and immortalized human microglia cultures will provide the scientific community with new and reliable tools for in vitro mechanistic studies of human microglia function in health from childhood to old age, and in disease, including our own studies of microglia function in Alzheimer's disease.