MOLECULAR STUDIES OF NK CELLS AND NK/LAK FUNCTION

NK 细胞和 NK/LAK 功能的分子研究

• 批准号: 3128427
• 负责人: FRITZ H BACH
• 金额: $ 17.83万
• 依托单位: NEW ENGLAND DEACONESS HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3128427
• 关键词: T lymphocyte; clone cells; gene expression; genetic library; histocompatibility antigens; human subject; laboratory mouse; laboratory rabbit; membrane proteins; molecular cloning; monoclonal antibody; natural killer cells; nucleic acid probes; transfection

The aim of this proposal is to identify cDNA clones corresponding to genes preferentially expressed in CD3- NK cells and other cells that express NK function and to understand the function of the corresponding gene products. A subtracted cDNA library enriched for cDNA clones associated with NK cells and NK/LAK activity will be prepared from a cloned NK cell (CD3-,CD16+,Leu19+) that expresses high-level NK/LAK function. A variety of screening methods will identify cDNA clones of interest within this library. Our first priority is to identify cDNA clones encoding cell surface molecules associated with NK cells and/or NK and LAK function. Second, we are interested in identifying clones encoding cell surface molecules expressed in both NK and T cells; this may identify molecules important in the regulation of NK function. Third, we are interested in clones that encode other molecules expressed preferentially in NK cells and other cells that express NK function. Two types of probes will be used to identify cDNA clones that encode membrane-bound components or secreted products. First, the subtracted library will be screened with a probe derived from poly-A+ RNA from membrane-bound polysomes of the cloned NK cell. Second, antisera prepared against the NK cell clone and absorbed with LCL will be used to probe the subtracted cDNA library prepared in a lambda-based expression vector. A long-term goal of this project is to use information and reagents obtained by molecular techniques to understand further the cell biology of NK cells, especially the generation and expression of NK/LAK activity. Whereas all genes to be studied will be derived from the NK clone described above, probing for expression of these genes in other cells, e.g. CD3+ cells with NK or NK and LAK function may help understand the genetic basis of these functions. Newly-defined genes will be sequenced and their expression studied in a variety of cell types; antisera/moAb will be made to proteins of interest. Although more difficult, we also plan to perform anti-sense (anti- mRNA) inhibition and transfection studies. We anticipate that these studies will provide information that will be useful as activated NK cells are used in clinical protocols.

该提案的目的是鉴定相应的 cDNA 克隆 CD3-NK细胞和其他细胞中优先表达的基因 表达 NK 功能并了解 NK 的功能 相应的基因产物。 消减 cDNA 文库富集 对于与 NK 细胞相关的 cDNA 克隆，NK/LAK 活性将 由克隆的 NK 细胞（CD3-、CD16+、Leu19+）制备而成 表达高级 NK/LAK 功能。 多种筛选 方法将识别该文库中感兴趣的 cDNA 克隆。 我们的首要任务是鉴定编码细胞表面的 cDNA 克隆 与 NK 细胞和/或 NK 和 LAK 功能相关的分子。 其次，我们有兴趣识别编码细胞的克隆 NK 和 T 细胞中表达的表面分子；这可能 鉴定在调节 NK 功能中重要的分子。 第三，我们对编码其他分子的克隆感兴趣 优先在 NK 细胞和其他表达 NK 功能。 两种类型的探针将用于鉴定 cDNA 编码膜结合成分或分泌产物的克隆。 首先，用衍生的探针筛选消减的文库 来自克隆 NK 膜结合多聚核糖体的聚 A+ RNA 细胞。 其次，针对NK细胞克隆制备抗血清并 用 LCL 吸收后用于探测消减的 cDNA 文库 在基于 lambda 的表达载体中制备。 长期目标 该项目的目的是使用获得的信息和试剂 分子技术进一步了解 NK 细胞生物学 细胞，尤其是 NK/LAK 活性的产生和表达。 而所有要研究的基因都来自 NK 克隆 如上所述，探测这些基因在其他细胞中的表达 细胞，例如 具有 NK 或 NK 和 LAK 功能的 CD3+ 细胞可能有帮助 了解这些功能的遗传基础。 新定义 基因将被测序并在多种中研究它们的表达 细胞类型；抗血清/单克隆抗体将针对感兴趣的蛋白质进行制备。 虽然比较困难，但我们也计划进行反义（anti- mRNA）抑制和转染研究。 我们预计 这些研究将提供有用的信息 活化的 NK 细胞用于临床方案。