Immunity in TSH-beta splice variant-deficient mice

TSH-β剪接变异缺陷小鼠的免疫

• 批准号: 8281950
• 负责人: JOHN R KLEIN
• 金额: $ 7.6万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-13 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8281950
• 关键词: Animals; Area; Bacterial Infections; Basal metabolic rate; Bone Marrow; Bone Marrow Cells; Bone Marrow Stem Cell; CD34 gene; Cell Separation; Cells; Disease; Endocrine; Endocrine system; Feasibility Studies; Future; Goals; Grant; Health; Hematopoietic; Hematopoietic stem cells; Hormones; Immune; Immune response; Immune system; Immunity; In Vitro; Infection; Inflammation; Laboratories; Lead; Lentivirus Vector; Leukocyte Trafficking; Metabolic; Metabolic Control; Mus; Outcome; Output; Protein Isoforms; Proteins; Protocols documentation; Publishing; RNA Splicing; Radiation Chimera; Recombinants; Regulation; Research; Research Project Grants; Research Proposals; Role; Source; Staining method; Stains; Subfamily lentivirinae; Testing; Thyroid Gland; Thyroid Hormones; Thyrotropin; Time; Variant; Virus Diseases; Work; base; cellular transduction; established cell line; in vitro testing; in vivo; meetings; novel; reconstitution; response; small hairpin RNA; trafficking; vector

DESCRIPTION (provided by applicant): There is growing evidence that the immune system is actively involved in metabolic regulation under both normal and pathophysiological conditions. Our laboratory recently identified a novel splice variant of the ¿-subunit of thyroid stimulating hormone (TSH¿) - the first functional TSH¿ isoform to be identified. Preliminary and published findings demonstrate that this splice variant is preferentially produced by cells of the immune system; that bone marrow (BM)-derived leukocytes traffic to the thyroid where they produce elevated levels of the splice variant in response to systemic virus infection; and that in vivo exposure of mice to recombinant splice variant protein curtails circulating thyroid hormone levels. Additionally, an shRNA that targets the TSH¿ splice variant blocks expression of the splice variant but not the native form of TSH¿. The goal of this R03 grant is to make mice in which the TSH¿ splice variant is suppressed in cells of the immune system. Preliminary studies using those animals will form the basis of a future R01 research proposal. There are two specific aims: Aim 1: To make a lentivirus/TSH¿- shRNA vector (LV-TSH¿-shRNA) that targets and suppresses the TSH¿ isoform. LV-TSH¿- shRNA vectors will be made using protocols that are familiar to our laboratory. These will be tested in vitro for their ability suppress the TSH¿ splice variant using established cell lines and in bone marrow hematopoietic cells. Aim 2 To make radiation chimeras using BM cells transduced with the LV-TSH¿-shRNA vector in order to suppress the splice variant in cells of the immune system. BM cells will be transduced with the LV-TSH¿-shRNA vector and enriched by cell sorting based on GFP expression (for identification of transduced cells) and CD34 expression (as a marker of early BM stem cells). These cells will be used to immunologically reconstitute irradiated syngeneic mice. Mice will be studied for their ability to respond to bacterial and viral infection. Findings from these studies will form the basi for a future R01 application aimed at understanding how the immune system contributes to the control of the metabolic response during infection and inflammation. PUBLIC HEALTH RELEVANCE: Although it has been known for many years that the immune system and the endocrine system interact collaboratively, the functional significance of this has remained obscure. In these studies, we will explore the mechanisms by the immune system regulates thyroid hormone levels using a recently identified new form of thyroid stimulating hormone.

描述（由申请人提供）：越来越多的证据表明，免疫系统在正常和病理生理条件下积极参与代谢调节。我们的实验室最近发现了一种新的剪接变体的亚基促甲状腺激素（TSH）-第一个功能性TSH？ 待鉴定的同种型。初步和已发表的研究结果表明，这种剪接变异体优先产生的免疫系统的细胞;骨髓（BM）来源的白细胞交通到甲状腺，在那里他们产生的剪接变异体的水平升高，以响应系统性病毒感染;和小鼠体内暴露于重组剪接变异体蛋白削减循环甲状腺激素水平。此外，靶向TSH剪接变体的shRNA阻断剪接变体的表达，但不阻断天然形式的TSH。这项R 03资助的目标是制造免疫系统细胞中TSH ²剪接变体受到抑制的小鼠。使用这些动物的初步研究将成为未来R 01研究提案的基础。有两个具体的目标：目标1：制备靶向和抑制TSH <$亚型的慢病毒/TSH <$- shRNA载体（LV-TSH <$-shRNA）。LV-TSH <$- shRNA载体将使用我们实验室熟悉的方案制备。将使用已建立的细胞系和骨髓造血细胞在体外测试它们抑制TSH剪接变体的能力。目的2利用转导LV-TSH <$-shRNA载体的BM细胞制备放射嵌合体，以抑制免疫系统细胞中的剪接变体。BM细胞将用LV-TSH-shRNA载体转导，并通过基于GFP表达（用于鉴定转导细胞）和CD 34表达（作为早期BM干细胞的标志物）的细胞分选进行富集。这些细胞将用于免疫重建辐射的同基因小鼠。将研究小鼠对细菌和病毒感染的反应能力。这些研究的结果将成为未来R 01应用的基础，旨在了解免疫系统如何有助于控制感染和炎症期间的代谢反应。 公共卫生关系：尽管免疫系统和内分泌系统协同作用已被人们所知多年，但其功能意义仍不清楚。在这些研究中，我们将探索免疫系统调节甲状腺激素水平的机制，使用最近发现的一种新形式的促甲状腺激素。