CHOLINERGIC NEURONS FOR TRANSPLANTATION

这个项目的总体目标是获得可以使用的细胞 用于移植以取代阿尔茨海默氏症和其他疾病中丢失的神经元 涉及胆碱能神经元的神经退行性疾病。两位将军 将研究各种方法；其中一种方法涉及转染人 关键的胆碱能蛋白进入能够获得神经元的细胞 表型，而另一种涉及使用来自 利用ChAT基因从胎儿神经细胞中筛选胆碱能神经元 含有胆碱能前体细胞的培养物。该项目涉及4个项目 明确的目标。第一个涉及细胞转导技术的使用。 为了介绍胆碱能细胞的三个主要组成部分， 胆碱转运体、胆碱乙酰转移酶和乙酰胆碱 转运蛋白进入能够获得神经元表型的细胞。这些 包括NT-2细胞、RN33B细胞和胚胎神经前体细胞。 包含胆碱能特定基因组序列的构建体将是 用于在胚胎胆碱能神经元中表达耐药基因 前体细胞。在药物存在的情况下生长将被用于 选择此群体中的胆碱能细胞。第二 特定的目标包括对被转染者或分离出的 细胞的胆碱能功能。这将集中在能力上 这些细胞在突触小泡中合成和储存乙酰胆碱， 去极化时释放乙酰胆碱。这些人的能力 与宿主神经元建立功能性相互作用的细胞将 最初通过将这些细胞与胎儿共同培养来进行体外检测 来自不同脑区的脑组织，寻找突触接触。 第三，将对转基因或分离的细胞进行测试，以检测其 大鼠脑内植入存活并继续存活的能力 合成、储存和分泌乙酰胆碱。最后，我们将确定 移植细胞改善特定行为的能力 基底前脑变性动物模型的缺陷。它是 预期这些研究将为进一步 可用于基因替换的神经细胞的发展 阿尔茨海默氏症及相关疾病的治疗。

The overall objective of this project is to obtain cells which can be used for transplantation to replace lost neurons in Alzheimer's and other neurodegenerative diseases involving cholinergic neurons. Two general approaches will be investigated; one involves transfecting genes of the key cholinergic proteins into cells capable of acquiring a neuronal phenotype, while the other involves the use of promoter elements from the ChAT gene to select out cholinergic neurons from fetal neuronal cell cultures containing cholinergic precursor cells. The project involves 4 specific aims. The first involves the use of cell transfection techniques to introduce the three major components of the cholinergic cell, the choline transporter, choline acetyltransferase, and the acetylcholine transporter into cells capable of acquiring a neuronal phenotype. These include NT-2 cells, RN33B cells, and embryonic neuronal precursor cells. Constructs containing the cholinergic specific genomic sequences will be used to express drug resistant genes in embryonic cholinergic neuronal precursor cells. Growth in the presence of the drug will be used to select for the cholinergic cells from this population. The second specific aim involves characterization of the transfected or isolated cells in terms of cholinergic function. This will focus on the ability of these cells to synthesize and store acetylcholine in synaptic vesicles, and to release acetylcholine upon depolarization. The ability of these cells to establish functional interactions with host neurons will initially be examined in vitro by co-culturing these cells with fetal brain tissue from various brain regions and looking for synaptic contacts. Thirdly, the transfected or isolated cells will be tested for their ability to survive intracerebral implantation in rats and to continue to synthesize, store, and secrete acetylcholine. Lastly, we will determine the ability of the implanted cells to ameliorate specific behavioral deficits in animal models of basal forebrain degeneration. It is anticipated that these studies will provide the basis for the further development of neuronal cells which can be used in gene replacement therapy for Alzheimer's and related diseases.