Regulation of circadian timers in a peripheral tissue the lung and identification of cellular and in vivo physiological pathways

该项目旨在发现昼夜节律计时器如何调节肺中的重要生理途径。已知许多疾病具有昼夜节律基础，在肺部，已知哮喘等炎症性疾病具有强烈的昼夜节律成分。此外，许多负责炎症反应的因子（细胞因子）也由生物钟驱动。我们将从定义肺中哪些细胞类型包含昼夜节律振荡器开始。这建立在我们的初步数据的基础上，这些数据表明，肺中可能存在特定的定时细胞，这些细胞也参与组织对外部损伤和炎症反应的反应。然后，我们将收集来自转基因小鼠的细胞，其中时钟基因启动子已适应驱动荧光素酶，并监测这些细胞在培养中发出的光，以跟踪潜在的生物钟。一旦我们建立了这些培养条件，我们将研究激素（糖皮质激素）如何重置昼夜节律振荡器的阶段。我们还将检查来自另一种小鼠品系的相同细胞，其中正常表达的肽受体（VPAC2）已被破坏。这只老鼠被认为是心律失常的，但据信外周组织中的昼夜节律振荡器仍然活跃，但不同步。通过培养这些细胞，我们的目标是看看它们是否能被糖皮质激素重新同步。一旦我们开发了这些方法，我们将继续研究由生物钟驱动的基因（所谓的生物钟控制基因）。我们的第一个候选基因是一个被称为CCAAT增强子结合蛋白或C/ ebp的基因家族，其中三个成员在肺中起作用。C/EBP很重要，因为它们可能驱动肺细胞中细胞因子活性的节律，从而导致昼夜炎症反应。我们将使用多种方法，包括用一种叫做siRNA的技术抑制C/EBP基因，然后看看我们是否可以阻断一种叫做白细胞介素-6的细胞因子基因的节律性活性，我们的初步数据已经显示白细胞介素-6是昼夜节律调节的。我们对活老鼠的研究将集中在两个问题上。首先，我们的目标是重新设置小鼠的肺时钟，每天用一种特殊配方的糖皮质激素喷雾（雾化）治疗它们一次，这样就只针对肺细胞。然后，我们将观察是否可以在正常和VPAC2突变小鼠中重新设置肺振荡器。接下来，我们将测试肺部对外部损伤的炎症反应的严重程度是否依赖于生物钟，在这项研究中，我们将使用正常小鼠和携带生物钟基因突变的动物（clk/clk小鼠），这种基因突变会导致个体昼夜节律振荡器不规律。我们需要区分肺部的昼夜节律计时器是否对这种反应有贡献，还是身体其他部位有其他贡献。为了测试这一点，我们的目标是重新设置肺时钟与身体其他部分的阶段，使用雾化糖皮质激素给两组动物在昼夜节律周期的相反阶段，相隔12小时。然后，我们会用外部刺激刺激动物，我们知道这会引起炎症反应，这样我们就可以测试组织反应的本质是否由定时系统控制。

This project aims to discover how circadian timers regulate an important physiological pathway in the lung. A number of diseases are known to have a circadian basis, and in the lung, inflammatory diseases such as asthma are known have a strong circadian component. In addition, many of the agents responsible for inflammatory responses (cytokines) are also driven by circadian clocks. We will start by defining which cell types in the lung contain circadian oscillators. This builds on our preliminary data which suggest that there may be specific timer cells in the lung which are also involved in tissue responses to external insult and inflammatory responses. We will then collect cells from a transgenic mouse in which a clock gene promoter has been adapted to drive luciferase, and monitor light emissions from these cells in culture to track the underlying circadian clock. Once we have established these culture conditions, we will examine how a hormone (glucocorticoid) resets the phase of the circadian oscillator. We will also examine the same cells from another mouse strain in which a normally expressed receptor for a peptide (VPAC2) has been disrupted. This mouse is known to be arrhythmic, but it is believed that circadian oscillators in peripheral tissues are still active, but unsynchronized. By culturing these cells, we aim to see whether they can be re-synchronised by glucocorticoids. Once we have developed these methods, we will proceed to study genes driven by the circadian clock (so-called clock controlled genes). Our first candidates are a family of genes called CCAAT enhancer binding proteins or C/EBPs, three members of which operate in the lung. C/EBP's are important as they may drive rhythms of cytokine activity in lung cells and hence circadian inflammatory responses. We will use a variety of methods including suppression of C/EBP genes with a technique called siRNA, and then seeing whether we can block the rhythmical activity of a cytokine gene called interleukin-6, which our preliminary data already shows is circadian regulated. Our studies on living mice will focus on two questions. First, we aim to re-set the lung clock in mice by treating them once a day with a specially formulated glucocorticoid in an aerosol spray (nebulised), so that only the lung cells are targeted. We will then see whether we can re-set lung oscillators in normal and VPAC2 mutant mice. We will next test whether the severity of the lung inflammatory responses to external insult is dependent on the circadian clock, and for this study we will use normal mice and animals bearing a mutation of the Clock gene (clk/clk mice) which renders individual circadian oscillators arrhythmic. We need to distinguish whether circadian timers in the lung contribute to the response or whether there other contributions from elsewhere in the body. In other to test this, we aim to re-set the phase of the lung clock from the rest of the body using nebulised glucorticoids given to two groups of animals at opposite phases of the circadian cycle, 12 hours apart. We will then challenge the animals with an external insult which we know will elicit an inflammatory response, so that we can test whether the nature of the tissue response is controlled by a timing system.