Regulation of Human TLR3 & TLR9 Expression and Function

人类 TLR3 的调控

• 批准号: 6668819
• 负责人: STEVEN L YOUNG
• 金额: $ 21.71万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6668819
• 关键词: cell line; clinical research; connective tissue cells; cytokine; endometrium; enzyme linked immunosorbent assay; epithelium; estradiol; female; hormone regulation /control mechanism; human subject; immunocytochemistry; progesterone; receptor expression; toll like receptor; western blottings

DESCRIPTION (provided by applicant): Cytokines are key regulators of two fundamental and distinct uterine functions: host defense and reproduction. The regulation of separate uterine functions by common cytokines results in cross-communication with significant impact on human health and disease. A shift in cytokine expression can serve the needs of host defense, but alter fetal tolerance contributing to infertility or pregnancy loss. Additionally, estrogen and progesterone can promote pregnancy initiation and maintenance by multiple means including modulation of cytokine expression resulting in altered host defense. To investigate the regulation of uterine cytokine expression, we have focused our attention on the endometrial epithelial cell, a prominent source of uterine cytokine expression and the first uterine cell type to contact an implanting embryo or ascending sperm or microbe. In non-uterine tissues, recognition of microbial molecules (and specific host molecules) by members of the Toll-like receptor (TLR) protein family can stimulate expression of cytokines. However, endometrial TLR expression and function remains unexplored. Preliminary data suggest two overall hypotheses: (1) TLRs stimulate expression of endometrial cytokines and (2) TLR-stimulated cytokine expression is modulated by estradiol and progesterone. In order to test these hypotheses, the proposed studies will focus on the endometrial expression and function of TLR3 and TLR9, which recognize nucleic acid motifs associated with viruses and bacteria, respectively. Initially, the endometrial cell types expressing TLR3 and TLR9 will be determined by immunohistochemical analysis of whole endometrium and real-time quantitative RT-PCR (qRT-PCR) analysis of separated epithelial and stromal cells taken from each phase of the menstrual cycle. The effects of estradiol and progesterone on expression of TLR3 and TLR9 expression in vitro will also be determined in primary endometrial cells as well as epithelial cell-lines (RL95-2 and Ishikawa) by qRT-PCR analysis. TLR3 and TLR9 function in endometrial cells and cell-lines will be assessed by measuring changes in cytokine expression after treatment with TLR3 and TLR9 ligands using ELISA and cytometric bead array (CBA) analyses. Finally, the intracellular signals utilized by endometrial TLRs in stimulating stimulate cytokine expression and their modulation by estradiol and progesterone will be investigated using pathway inhibitors and western blot analysis. Thus, the proposed studies will contribute to an understanding of mechanisms regulating human endometrial cytokine expression, which could lead to the design of pharmacologic therapies for both infectious and reproductive disorders.

描述（由申请人提供）：细胞因子是两种基本而独特的子宫功能：宿主防御和生殖的关键调节因子。共同细胞因子对不同子宫功能的调控产生交叉交流，对人类健康和疾病有重要影响。细胞因子表达的改变可以满足宿主防御的需要，但会改变胎儿的耐受性，导致不孕或妊娠丢失。此外，雌激素和孕激素可以通过多种方式促进妊娠的开始和维持，包括调节细胞因子的表达，从而改变宿主的防御。为了研究子宫细胞因子表达的调控，我们将注意力集中在子宫内膜上皮细胞上，它是子宫细胞因子表达的重要来源，也是第一个与着床胚胎或上升精子或微生物接触的子宫细胞类型。在非子宫组织中，toll样受体（TLR）蛋白家族成员对微生物分子（和特定宿主分子）的识别可以刺激细胞因子的表达。然而，子宫内膜TLR的表达和功能尚不清楚。初步数据提出两种假说：(1)tlr刺激子宫内膜细胞因子表达；(2)tlr刺激的细胞因子表达受雌二醇和黄体酮调节。为了验证这些假设，我们将重点研究TLR3和TLR9的子宫内膜表达和功能，它们分别识别与病毒和细菌相关的核酸基序。最初，将通过对整个子宫内膜的免疫组织化学分析和对月经周期各阶段分离的上皮细胞和基质细胞的实时定量RT-PCR （qRT-PCR）分析来确定表达TLR3和TLR9的子宫内膜细胞类型。在体外实验中，我们还将通过qRT-PCR分析雌二醇和孕酮对子宫内膜原代细胞以及上皮细胞系（RL95-2和Ishikawa） TLR3和TLR9表达的影响。TLR3和TLR9在子宫内膜细胞和细胞系中的功能将通过使用ELISA和细胞头阵列（CBA）分析测量TLR3和TLR9配体处理后细胞因子表达的变化来评估。最后，我们将使用途径抑制剂和western blot分析来研究子宫内膜tlr在刺激细胞因子表达以及雌二醇和黄体酮对细胞因子表达的调节中所利用的细胞内信号。因此，所提出的研究将有助于理解调节人类子宫内膜细胞因子表达的机制，这可能导致设计用于感染性和生殖疾病的药物治疗。