Molecular Basis of Human VH Gene Replacement

描述(申请人提供)：应聘者是一名分子生物学家 希望在分子免疫学方面建立独立的研究生涯 菲尔德。在马克斯·D·库珀？S博士的指导下进行的免疫学强化培训是 建议在最初的两到三年内完成研究 在授权期内保持独立性。该提案将重点放在分子 人类VH基因替换的基础、调控机制及其意义 对正常B细胞发育、B细胞反应和类风湿的贡献 尤其是关节炎。这一假说建立在对克隆人的分析基础上 从Prob体外连续分化的细胞系EU12 (CD34+)转化为Preb(CD34 mU SLC+)，然后转化为B细胞(CD34 mU+LC)并产生 通过系列VH替换实现无性系内多样性。正在进行的VH 通过检测VH替换来验证EU12单元中的替换 在隐秘的RSS位点出现切除圈和双链DNA断裂。(目标 1)以EU12细胞为实验模型，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，以确定隐蔽的功能 RSS站点。(目标2)使用从研究中得出的方法和原则 Eu12模型，正常人骨髓和扁桃体生发中心B 将利用系谱细胞研究VH的发生和分期(S) 在人类身上进行替换。(目标3)VH更换的监管机制将 使用EU12模型通过评估的效果进行调查。(I) 调制替代轻链(Lamda 5/14.1)或常规轻链 (3)结合细胞表面受体(前BCR， BCR和CD40)，以及(Iii)用细胞因子(IL 1β、TFNAF1a、IL)刺激 6、IL 7)VH置换。(目标4)确定VH更换是否发生在 滑膜B细胞因此有助于产生自身抗体和 寻找类风湿性关节炎滑膜中潜在的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.