CORE--PATHOGENESIS AND PREVENTION OF TYPE I DIABETES IN THE NOD MOUSE AND MAN

这个核心单元将提供复杂的三色免疫荧光 通过提供专用的细胞分析和三色细胞分选 荧光激活细胞分选机和专职技术操作员，以 支持构成以下基础的五个合作研究项目 本研究计划项目建议书。每个协作项目 在这个方案中严重依赖于使用荧光激活的细胞 分析和细胞分选以实现他们的实验目标。因此，Dr。 钱学森利用Dr。 McDevitt将开发生产多价特定产品的技术 可检测各种T细胞的多肽/I-/A/G7染色试剂 特定多肽/I-A/G7复合体的特异性细胞出现在 NOD小鼠IDDM的发生发展过程。戴维斯博士，在 与钱博士的合作开发了聚合酶链技术 14-18日龄NOD小鼠单个T细胞的反应分析 这导致了T细胞利用的序列的确定 这些T细胞中的受体，并最终将导致产生 表达胰岛素致炎T细胞受体的转基因小鼠获得表达 在I型IDDM病程的早期。这个项目需要高精尖的技术 细胞分选以剔除胰岛制剂中的所有外来细胞 为了分离这些胰岛中数量相对较少的T细胞 在糖尿病发生过程的最开始。克拉布特里博士，在 与麦克德维特博士的合作将培育出表达突变的NOD小鼠 可阻断T细胞受体信号转导通路的亲环素 NOD小鼠发生糖尿病的不同时间点。 荧光激活细胞分选(FACS)分析将表征 在这些免疫抑制和胰岛细胞中产生的T细胞群 耐受抗原的动物。类似地，由Dr。 Weissman和Shizuru将需要复杂的细胞分类来分离 用于干细胞移植到幼年NOD小鼠体内的造血干细胞， 利用H2相合的干细胞和全骨髓 同基因捐献者。中国人T细胞受体谱系的研究进展 这只小鼠将使用Dr。 钱，对T细胞受体序列的单细胞分析 钱博士和戴维斯博士以及表征T细胞的方法 克拉布特里博士使用的受体曲目。

This core unit will provide sophisticated three color immunofluorescent cell analysis and three color cell sorting by providing a dedicated fluorescence activated cell sorter, and full time technical operator, to support the five collaborative research projects which form the basis of this research program project proposal. Each of the collaborative projects in this program rely heavily on the use of fluorescence activated cell analysis and cell sorting to achieve their experimental goals. Thus, Dr. Chien utilizing data on dominant peptide epitopes provided by Dr. McDevitt, will develop techniques for production of polyvalent specific peptide/I-/A/g7 staining reagents which can detect all of the various T cells specific for a particular peptide/I-A/g7 complex appearing in the course of the development of IDDM in the NOD mouse. Dr. Davis, in collaboration with Dr. Chien has developed techniques for polymerase chain reaction analysis of single T cells isolated from 14-18 day old NOD mice. This has lead to determination of sequences utilized by the T cell receptors in these T cells, and ultimately will lead to the production of transgenic mice expressing insulitis-inducing T cell receptors expressed early in the course of type I IDDM. This project requires sophisticated cell sorting to gate out all of the extraneous cells in islet preparations to isolate the relatively small number of T cells present in these islets at the very beginning of the diabetogenic process. Dr. Crabtree, in collaboration with Dr. McDevit will produce NOD mice expressing mutant cyclophilins which will permit blockade of T cell receptor signaling at various time points in the development of diabetes in NOD mice. Fluorescent Activated Cell Sorting (FACS) analysis will characterize the resultant T cell populations in these immunosupressed and islet cell antigen tolerant animals. Similarly, the experiments proposed by Dr. Weissman and Shizuru will require sophisticated cell sorting to isolate hematopoietic stem cell for stem cell transplantation into young NOD mice, utilizing stem cells and whole bone marrow from H2-matched as well as syngeneic donors. The development of the T cell receptor repertoire in this mice will be monitored using the staining reagents developed by Dr. Chien, the single cell analysis of T cell receptor sequences developed by Drs. Chien and Davis, and the methods for characterizing the T cell receptors repertoire utilized by Dr. Crabtree.