Molecular Basis of Human VH Gene Replacement

描述（由申请人提供）：候选人是分子生物学家， 希望在分子免疫学领域建立独立的研究事业 领域在Max D博士的指导下进行培训。库珀？在免疫学方面的监督是 建议在最初的两三年内， 在授标期间保持独立性。该提案将侧重于分子 人VH基因置换的基础和调控机制及其对人类免疫功能的影响 促进正常B细胞发育、B细胞应答和类风湿性关节炎 尤其是关节炎。该假说建立在对克隆人的分析基础上， 细胞系，EU 12，经历proB的连续体外分化 (CD34+）与前B（CD 34 mu SLC+），然后与B细胞（CD 34 mu+LC），并产生 通过连续VH置换的克隆内多样性。正在进行的VH 通过检测VH置换来验证EU 12细胞中的VH置换 切除环和双链DNA在隐蔽的RSS位点处断裂。（目标 1)EU 12细胞将被用作实验模型，以解剖 VH置换的分子基础。纯化的RAG 1、RAG 2和HMG 1 蛋白质将用于体外研究，以确定隐藏的功能， RSS站点。(Aim（2）运用从研究中得出的方法和原则， EU 12模型、正常人骨髓和扁桃体生发中心B 谱系细胞将用于研究VH的发生和阶段 人类的替代品。(Aim 3）VH替代的调节机制将 使用欧盟12模型进行研究，通过评估的影响。（一） 调节替代轻链（λ 5/14. 1)或常规轻链 （κ和λ X）表达，（iii）连接细胞表面受体（前BCR， BCR和CD 40），和（iii）用细胞因子（IL 1 β，TFNaphla，IL 6和IL 7）对VH置换的影响。(Aim 4）确定VH置换是否发生在 滑膜B细胞，从而有助于自身抗体的产生， 在类风湿关节炎滑膜中寻找潜在的VH替代刺激， 组织.

DESCRIPTION(provided by applicant): The candidate is a molecular biologist who wishes to establish an independent research career in the molecular immunology field. Enhanced training under Dr. Max D. Cooper?s supervision in immunology is proposed for the initial two or three years in order to achieve research independence within the award period. The proposal will focus on the molecular basis and regulatory mechanism of human VH gene replacement and its contribution to normal B cell development, B cell responses, and rheumatoid arthritis in particular. The hypothesis builds on an analysis of a clonal human cell line, EU12, that undergoes continuous in vitro differentiation from proB (CD34+) to preB (CD34 mu SLC+) then to B cells (CD34 mu+LC) and generates intraclonal diversity through serial VH replacements. The ongoing VH replacement in the EU12 cells is verified by the detection of VH replacement excision circles and double stranded DNA breaks at the cryptic RSS sites. (Aim 1) The EU12 cells will be used as an experimental model to dissect the molecular basis for the VH replacement. Purified RAG 1, RAG 2, and HMG 1 proteins will be used in in vitro studies to define the function of the cryptic RSS site. (Aim 2) Using the methods and principles derived from the study of the EU 12 model, normal human bone marrow and tonsillar germinal center B lineage cells will be used to investigate the occurrence and stage(s) of VH replacement in humans. (Aim 3) The regulatory mechanism of VH replacement will be investigated using the EU12 model through evaluating the effects of. (i) modulating surrogate light chain (lamda 5/14. 1) or conventional light chain (kappa and lamdaX) expression, (iii) ligating cell surface receptors (pre BCR, BCR, and CD40), and (iii) stimulating with cytokines (IL 1 beta, TFNaphla, IL 6, and IL 7) on VH replacement. (Aim 4) Determine if VH replacement occurs in synovial B cells thereby contributing to the generation of autoantibodies and to search for potential VH replacement stimuli in rheumatoid arthritis synovial tissue.