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

这个项目旨在发现昼夜节律计时器如何调节肺部的一条重要的生理通路。已知许多疾病具有昼夜节律基础，而在肺部，已知哮喘等炎症性疾病具有很强的昼夜节律成分。此外，许多负责炎症反应(细胞因子)的因素也是由生物钟驱动的。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基础上，这些数据表明，肺中可能存在特定的计时细胞，这些细胞也参与组织对外部侮辱和炎症反应的反应。然后，我们将从时钟基因启动子已被适应来驱动荧光素酶的转基因小鼠中收集细胞，并监测这些细胞在培养中的光发射，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相位。我们还将检查来自另一种小鼠品系的相同细胞，在该品系中，正常表达的多肽受体(VPAC2)已被破坏。众所周知，这种小鼠是心律不齐的，但人们认为，周围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可以被糖皮质激素重新同步。一旦我们开发出这些方法，我们将继续研究由生物钟驱动的基因(所谓的时钟控制基因)。我们的第一批候选基因是一个名为CCAAT增强子结合蛋白或C/EBPs的基因家族，其中三个成员在肺中工作。C/EBP很重要，因为它们可以驱动肺细胞中细胞因子活动的节律，从而导致昼夜节律的炎症反应。我们将使用各种方法，包括用一种名为siRNA的技术抑制C/EBP基因，然后看看我们是否能阻断一种名为白细胞介素6的细胞因子基因的节律性活动，我们的初步数据已经表明，白介素6是受昼夜节律调节的。我们对活老鼠的研究将集中在两个问题上。首先，我们的目标是通过在雾化喷雾剂中每天给小鼠一次特殊配方的糖皮质激素来重新设置小鼠的肺时钟，这样就只有肺细胞成为靶点。然后，我们将看看是否可以在正常和VPAC2突变小鼠身上重新设置肺振荡器。接下来，我们将测试肺部对外部刺激的炎症反应的严重程度是否取决于生物钟，在这项研究中，我们将使用正常小鼠和携带时钟基因突变的动物(CLK/CLK小鼠)，这种突变使单个昼夜节律振荡器心律失常。我们需要区分肺中的昼夜节律计时器是否参与了这种反应，或者是否还有来自身体其他部位的其他贡献。为了测试这一点，我们的目标是使用雾化吸入的糖皮质激素来重新设置肺部时钟与身体其他部分的相位，这两组动物处于昼夜周期的相反阶段，相隔12小时。然后，我们将用外部侮辱来挑战动物，我们知道这会引发炎症反应，这样我们就可以测试组织反应的性质是否由计时系统控制。

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.