HUMAN B CELL DIFFERENTIATION IN A MODEL SYSTEM

模型系统中的人类 B 细胞分化

• 批准号: 2886969
• 负责人: LORI Ruth COVEY
• 金额: $ 11.87万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-15 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2886969
• 关键词: B lymphocyte; CD40 molecule; cell differentiation; cytokine; gene mutation; gene rearrangement; genetic transcription; human genetic material tag; human tissue; immunoglobulin genes; leukocyte activation /transformation; polymerase chain reaction; tissue /cell culture; B lymphocyte; CD40 molecule; cell differentiation; cytokine; gene mutation; gene rearrangement; genetic transcription; human genetic material tag; human tissue; immunoglobulin genes; leukocyte activation /transformation; polymerase chain reaction; tissue /cell culture

The overall goal of this is to understand the regulation of immunoglobulin class switch recombination in human B cells. Specifically, we will use a model system that is comprised of a human B cell lymphoma cell line, RAMOS 266, that has the capacity to undergo isotype differentiation in response to cytokine and T cell-mediated signals. In addition, it was found that initial activation of the RAMOS B cells could be measured by up-regulation of surface CD23 and was dependent on the CD40 ligand (CD40-L) expressed on the surface of the D1.1 T cells. We will take advantage of RAMOS 266's capacity to undergo differentiation in culture to assess the transcriptional activation of the different constant region genes in response to cytokines and T cell help. In this project, T cell help will be defined as signaling through the CD40-L. Initial experiments will be carried out using RT-PCR on RAMOS mRNA isolated after exposure to CD40-L and/or cytokines. We will assess the response of the different isotype classes and subclasses to these signals and evaluate the response in terms of establishing whether class switching is proceeding by a directed or stochastic process. Our analyses will include measuring the germline (I- CH) transcript expression as well as the expression of mature (VDJ-CH) transcripts. To study more completely the regulation of isotype switching, we will analyze switch variants of RAMOS 266 cells and determine their capacity for further switch events. Additionally, we plan to evaluate the rearrangement events and the transcriptional response on the non- productive alleles of the switch variants to further establish a mechanism for the regulation of class switch. And finally, we will mutate the I gamma I exon of the heavy chain C gamma 1 region using homologous recombination. We will identify the rearrangement status of the non- productive allele after simulation with cytokines and T cell contact.

总体目标是了解免疫球蛋白的调节 人类B细胞中的类开关重组。 具体来说，我们将使用 由人B细胞淋巴瘤细胞系组成的模型系统Ramos 266，有能力进行同种型分化以响应 到细胞因子和T细胞介导的信号。此外，发现 可以通过上调来测量Ramos B细胞的初始激活 表面CD23的表面，取决于表达的CD40配体（CD40-L） D1.1 T细胞的表面。我们将利用Ramos 266的优势 在文化中差异化的能力来评估 不同常数区域基因的转录激活 对细胞因子和T细胞帮助的反应。在这个项目中，T细胞帮助将 通过CD40-L定义为信号。最初的实验将是 暴露于CD40-L后，使用RT-PCR对RAMOS mRNA进行了 和/或细胞因子。我们将评估不同同种型的响应 这些信号的课程和子类，并评估响应 确定班级切换是由指导或 随机过程。我们的分析将包括测量种系（i- CH）转录本表达以及成熟的表达（VDJ-CH） 成绩单。为了更彻底地研究同种型切换的调节， 我们将分析Ramos 266个细胞的开关变体，并确定它们 进一步切换事件的能力。此外，我们计划评估 重新排列事件和非 - 非转录响应 开关变体的生产性等位基因以进一步建立机制 用于班级开关的调节。最后，我们将变异我 使用同源 重组。我们将确定非 - 用细胞因子和T细胞接触模拟后的生产性等位基因。