NK CELL GENESIS FROM HUMAN BONE MARROW PROGENITORS

自然杀伤(NK)细胞在抗肿瘤和抗肿瘤中发挥重要作用 抗微生物防御，在淋巴和造血调节中 细胞，在生产各种细胞因子，并代表 淋巴因子激活的杀伤细胞中的优势效应细胞 (LAK)现象。尽管NK具有生物学和临床意义 细胞，对定义NK细胞谱系的关注很少 以及了解NK细胞发育的成熟事件。我们的 该提案旨在弥合这一差距。现在可以研究自然语言了 细胞谱系和发育的最新进展 重组DNA技术、分子方法和可获得性 过多的单抗、细胞因子和遗传探针。这个 我们调查的具体目的是研究：(1)NK的产生 人骨髓CD34祖细胞及其亚群的细胞， 包括造血干细胞(HSC)，使用长期骨髓 LTBMC培养系统；(2)NK细胞的逐步鉴定 LTBMC分化事件与NK细胞克隆分析 祖细胞；LTBMC将被分析：形态和获取 成熟期间的细胞质和细胞表面抗原，使用两种和 三色流式细胞术：不同形态NK细胞的表达 肿瘤识别结构(NK-TR)，由交替的mRNA决定 剪接，使用聚合酶链式反应技术；zeta亚基，使用细胞渗透性 程序；NK祖细胞的频率，使用有限稀释法； 将尝试制备抗NK细胞的单抗 祖先。(3)NK细胞的细胞毒性和发育过程中的事件 LTBMC的调节功能，包括NK细胞细胞毒的形成 机制、结合/裂解活性的获得、颗粒酶和细胞溶血素 系统，产生细胞因子和调节造血的能力。这 NK细胞成熟的逐步分析，从最多开始 原始干细胞舱，将让我们第一次“作图” 时间、NK分化途径(S)。这些信息将有助于 回答关于NK细胞谱系的问题，将有助于 确定一些成熟途径涉及到的获取 NK细胞的生物学功能。通过“映射”正常的NK细胞成熟-- ，则有可能检测到不同的分化途径 或癌症患者的非同步结构/功能关系， 尤其是白血病。

Natural killer (NK) cells play an important function in antitumor and anti-microbial defense, in regulation of lymphoid and hematopoietic cells, in production of a variety of cytokines, and represent the dominant effector cells involved in lymphokine-activated killer cell (LAK) phenomenon. Despite the biological and clinical significance of NK cells, very little attention has been given to defining NK cell lineage and understanding the maturational events in NK cell development. Our proposal is aimed at bridging this gap. It is now possible to study NK cells lineage and development as a result of the recent advances in recombinant DNA technology, molecular approaches and availability of a plethora of monoclonal antibodies, cytokines and genetic probes. The specific aims of our investigations are to study: (1) Generation of NK cells from human bone marrow CD34+ progenitors and their subsets, including hematopoietic stem cells (HSC), using long-term bone marrow culture (LTBMC) systems; (2) Stepwise characterization of NK cell differentiation events in LTBMC and subsequent clonal analysis of NK cell progenitors; LTBMC will be analyzed for: morphology and for acquisition of cytoplasmic and cell surface antigens during maturation, using two and three-color flow cytometry; expression of different forms of NK cell tumor recognition structure (NK-TR), as determined by alternate mRNA splicing, using PCR technique; zeta subunit, using cell-permeabilization procedure; frequency of NK progenitors, using limiting dilution assay; an attempt will be made to prepare monoclonal antibodies against NK cell progenitors. (3) Events in development of NK cell cytotoxic and regulatory functions in LTBMC, including development of NK cell cytotoxic mechanism, acquisition of binding/lytic activity, granzyme and cytolysin systems, ability to produce cytokines and regulate hematopoiesis. This step-wise analysis of NK cell maturation, starting with the most primitive stem cell compartment, will allow us to "map", for the first time, NK differentiation pathway(s). This information will help to answer the questions of NK cell lineage and will be instrumental in determining the maturational pathways involved in acquisition of some of the NK cell biological functions. By "mapping" of normal NK cell matura- tion, it may be possible to detect divergent pathways of differentiation or asynchronous structure/function relationships in cancer patients, especially leukemia.