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: 3477795
• 负责人: DEBORAH HARBOUR
• 金额: $ 8.86万
• 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/3477795
• 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.

免疫系统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介导， 受体。具体地说，免疫系统既产生又响应 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 并表征了体外T细胞模型（Molt 4淋巴母细胞），其 对促甲状腺激素释放刺激作出反应， 激素（TRH）。此外，这些细胞还表达细胞表面结合 TRH的位点，该提案的总体目标是生物化学和 分子表征蜕皮产生的TSH和TRH受体 4个T细胞。这一信息将回答Molt 4细胞模型是否 促甲状腺细胞产生垂体TSH的精确模型。的 实验将包括测定Molt 4细胞的生物活性 神经内分泌系统的细胞上的衍生的TSH，以及对细胞的 免疫系统.将在RNA水平上分析ir-TSH， 确定TSH和β的基因是否在同一细胞中表达， 动力学方式，并使用相同数量和类型的TSH mRNA作为 腐败TSH蛋白和mRNA的调节将在 对垂体源性TSH的初级和次级调节剂的反应。在 此外，将测定来自经典免疫系统的细胞因子， 他们调节ir-TSH的能力。本研究结果可为体外研究提供参考 与体内if-TSH产生的作用的相关性 免疫反应这些实验将决定 基因的表达和调节使用与那些使用类似的途径， 垂体系统这些数据将确定Molt 4细胞是否 模型是一个代表性的模型，用于研究调控机制， HLT轴可能类似于 HPT轴。此外，这些数据将为以下途径提供信息： 免疫系统的独特调节。Molt 4细胞 似乎有两个TRH结合位点，一个低亲和力位点类似于 垂体TRH受体和一个高亲和力位点，这是独特的， 免疫系统该项目建议对这些结合位点进行表征， 生物化学和关于第二信使途径。结果 将这些实验的结果与GH 3大鼠上的TRH受体进行比较 垂体细胞TRH受体的结合链将是 测定、纯化并用于生产单克隆抗体。 该抗体将用作筛选λ gt 11 cDNA的试剂 图书馆最后，我们建议将cDNA克隆到 Molt 4细胞上的TRH受体。发展和 Molt 4细胞体外模型的表征和 它作为研究TSK和TRH基因的系统的有用性 从基础和临床科学的观点来看，受体是重要的。 目前还没有稳定的体外细胞系作为一个完整的 TSH产生的模型，因此是HPT或HLT的模型 轴线因此，该模型可以证明是非常有用的分析和 基因的结构和调控的表征， 人TSH α和β的启动子。此外， HLTT和HPT轴的调节可以使用该模型来解决。 单克隆抗体的纯化、制备及克隆 TRH受体将是第一次， 在免疫系统上表达的神经内分泌释放激素已经被 克隆的这些分析将有助于开发试剂， 探测HPT和HLT轴。从临床科学的角度来看，这些 试剂可以作为探针，用于研究具有缺陷的个体， HPT和/或HLT轴。