IN VITRO NEUROENDOCRINE T CELL LINE PRODUCTION OF TSH

体外神经内分泌 T 细胞系生产 TSH

• 批准号: 3477796
• 负责人: DEBORAH HARBOUR
• 金额: $ 8.07万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-30 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3477796
• 关键词: RNA; T lymphocyte; biological models; complementary DNA; gene expression; genetic regulation; hormone receptor; laboratory rat; membrane activity; messenger RNA; molecular cloning; monoclonal antibody; neuroendocrine system; receptor binding; second messengers; thyrotropin; thyrotropin releasing hormone; tissue /cell culture

The immune and neuroendocrine system interact through communication mediated on a molecular basis by the production of similar ligands and receptors. Specifically, the immune system both produces and responds to thyrotropin (TSH). We have postulated that there exists a hypothalamic- lymphoic-thyroid (HLT) axis. We have identified and partially developed and characterized an in vitro T cell model (Molt 4 lymphoblast) which expresses thyrotropin in response to stimulus with thyrotropin releasing hormone (TRH). In addition, these cells also express cell surface binding sites for TRH., The overall goal of this proposal is to biochemically and molecularly characterize the TSH and TRH receptor(s) produced by the Molt 4 T cells. This information will answer whether the Molt 4 cell model is an accurate model of pituitary TSH production by thyrotroph cells. The experiments will include determination of bioactivity of the Molt 4 cell derived TSH on cells of the neuroendocrine system as well as on cells of the immune system. The ir-TSH will be analyzed at the level of RNA to determine whether the genes for TSH and beta are expressed in the same kinetic fashion, and use the same number and types of TSH mRNAs as the putuitary. The reulation of TSH protein and mRNA will be analyzed in response to primary and secondary regulators of pituitary derived TSH. In addition, cytotines from the classical immune system will be assayed for their ability to regulate the ir-TSH. The results may provide in vitro relevance to the role of if-TSH production in vivo in the mileau of an immune response. These type of experiments will determine whether the genes are expressed and regulated using similar pathways as those used by the pituitary system. These data will define whether the Molt 4 cell model is a representative model for studying mechanisms of regulation of the HLT axis that may be similar to the mechanisms of regulation of the HPT axis. Additionally, the data will lend information to pathways of regulation that may be unique to the immune system. The Molt 4 cells appear to have two binding sites for TRH, a low affinity site similar to the pituitary TRH receptor and a high affinity site that is unique to the immune system. This project proposes to characterize these binding sites, biochemically and with regards to second messenger pathways. The results of these experiments will be compared to the TRH receptors on GH3 rat pituitary cells. The binding chain(s) of the TRH receptor(s) will be determined, purified and used for production of a monoclonal antibody. The antibody will be used as a reagent to screen a lambda gt11 cDNA library. Finally, we propose to clone the cDNA to the binding chain of the TRH receptor(s) on the Molt 4 cells. The development and characterization of the Molt 4 cell in vitro model and verification of its usefulness as a system for studying the genes for TSK and TRH receptors is important from a basic and clinical science standpoint. There is currently no stable in vitro cell line that serves as a complete model for TSH production and is therefore a model for the HPT or HLT axis. Therefore, this model may prove very useful for analysis and characterization of the structure and regulation of the genes and promoters for human TSH alpha and beta. In addition, mechanisms of regulation of the HLTT and HPT axis may be addressed using this model. The purification, development of a monoclonal antibody and cloning of the TRH receptor(s) will be the first time that a receptor for a neuroendocrine releasing hormone expressed on the immune system has been cloned. These analyses will be useful in the development of reagents for probing the HPT and HLT axis. from a clinical science standpoint, these reagents may serve as probes in studying individuals with defects in the HPT and/or HLT axis.

免疫和神经内分泌系统通过交流相互作用。 在分子基础上通过产生类似的配体和 感受器。具体地说，免疫系统既产生并响应 促甲状腺激素(TSH)。我们假设存在一个下丘脑- 淋巴甲状腺(HLT)轴。我们已经确定并部分开发了 并鉴定了一种体外T细胞模型(Molt 4淋巴母细胞)，该模型 通过释放促甲状腺激素来响应刺激而表达促甲状腺激素 激素(TRH)。此外，这些细胞还表达细胞表面结合。 这项提议的总体目标是在生物化学和 蜕皮产生TSH和TRH受体(S)的分子特征 4 T细胞。此信息将回答Molt 4细胞模型是否 促甲状腺细胞分泌TSH的准确模型。这个 实验将包括测定Molt 4细胞的生物活性。 促性腺激素对神经内分泌系统细胞和小鼠神经内分泌细胞的影响 免疫系统。Ir-TSH将在RNA水平上进行分析，以 确定促甲状腺激素基因和β基因是否在相同的 动态时尚，并使用相同数量和类型的TSH mRNA作为 公证的。TSH蛋白和信使核糖核酸的调节将在 对垂体源性TSH初级和次级调节剂的反应。在……里面 此外，来自经典免疫系统的胞嘧啶将被检测 它们调节ir-TSH的能力。该结果可能会在体外提供 与体内IF-TSH产生在AN环境中的作用的相关性 免疫反应。这些类型的实验将决定 基因的表达和调控使用的途径与 脑下垂体系统。这些数据将定义Molt 4细胞 模型是研究植物生长调节机制的代表性模型。 HLT轴可能类似于HLT的调节机制 HPT轴。此外，这些数据将把信息提供给 这可能是免疫系统独有的调节。Molt 4细胞 似乎有两个TRH的结合位点，一个类似于 垂体TRH受体和一个高亲和力部位，该部位是 免疫系统。该项目建议对这些结合位点进行表征， 生化和与第二信使途径有关的。结果是 这些实验将与GH3大鼠的TRH受体进行比较 脑垂体细胞。TRH受体(S)的结合链(S)将是 测定、提纯并用于生产单抗。 该抗体将作为筛选lambda gt11基因的试剂。 图书馆。最后，我们建议将该基因克隆到其结合链上。 Molt 4细胞上的促肾上腺皮质激素释放激素受体(S)。中国的发展和 Molt-4细胞体外模型的表征及模型的验证 它作为研究TSK和TRH基因的有用系统 从基础和临床科学的角度来看，受体是重要的。 目前还没有稳定的体外细胞系作为完整的 TSH生产模型，因此是HPT或HLT的模型 轴心。因此，这个模型可能会被证明是非常有用的分析和 基因和调控基因的结构和调控的特征 人类促甲状腺激素α和β的启动子。此外，该机制还包括 HLTT和HPT轴的调节可以使用该模型来解决。 单抗的纯化、制备及克隆 促肾上腺皮质激素受体(S)将是一种受体首次用于 表达在免疫系统上的神经内分泌释放激素已经 克隆的。这些分析将有助于开发新的试剂。 探测HPT和HLT轴。从临床科学的角度来看，这些 试剂可作为研究有缺陷的个体的探针。 HPT和/或HLT轴。