IN VITRO REAL-TIME CIRCADIAN RHYTHM MEASUREMENT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 昼夜节律产生由生理调节的大约24小时节律模式组成，其中表达的适当调节对于维持良好健康至关重要。昼夜节律产生的紊乱似乎与许多疾病有关，如季节性情感障碍、各种其他精神疾病、睡眠问题、癌症和许多代谢综合征。昼夜节律紊乱可能在其中几种疾病的发病机制中具有某种致病作用，因为有证据表明与异常昼夜节律产生相关的遗传因素与双相情感障碍、家族性睡眠相位提前综合征和睡眠相位延迟综合征等疾病之间存在联系。 尽管在人类中已经报道了昼夜节律紊乱和几种疾病之间的关联，但昼夜节律产生影响疾病表现的确切机制对于许多疾病和病症仍然是未知的。限制因素之一是与人体昼夜节律表达的生理测量相关的技术困难。为了更好地了解患者的昼夜节律产生、昼夜节律紊乱在发病机制中的意义以及评估潜在的药物治疗，重要的是开发一种非侵入性、简单、快速和可靠的体外测定方法来确定人体的昼夜节律。 最近的研究结果使研究人员能够证明使用外周细胞来分析人体昼夜节律的可行性。到目前为止，真皮成纤维细胞是唯一的外周细胞，其中稳定的昼夜节律可以记录在人类。然而，皮肤活检并不是大多数临床部门的常规程序;它们对患者来说也是痛苦的，并且经常留下疤痕。因此，开发使用一些其它细胞类型（即，血细胞）。 在本提案中，我们的目标是开发一种非侵入性的体外测定系统来测量来自人血液样品的巨噬细胞中的人昼夜节律，使用时钟基因报告子实时监测昼夜节律参数（总共将收集31名健康受试者）。一旦建立了测定系统，该技术将被应用于评估化学品的影响，从而测试该测定作为未来诊断工具的可行性（共将招募30名参与者）。 CRC将用于招募健康志愿者并采集血液样本用于上述目的。将要求每名受试者捐献5 mL血液两次，至少间隔一个月。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Circadian rhythm generation consists of an approximate 24 h rhythmic pattern of physiologic regulation whereby proper regulation of the expression is critical to maintaining good health. Disturbances in circadian rhythm generation appear to be associated with many diseases such as seasonal affective disorders, a variety of other mental illnesses, sleep problems, cancer, and a number of metabolic syndromes. It is possible that circadian disruptions have some causative role in the pathogenesis of several of these diseases, as there is evidence suggesting links between genetic factors related to abnormal circadian rhythm generation and illnesses like bipolar affective disorder, familial advanced sleep phase syndrome, and delayed sleep phase syndrome. Even though associations between circadian rhythm disturbances and several diseases have been reported in humans, the precise mechanisms by which circadian rhythm generation impacts the manifestation of disease remains unknown for many illnesses and disorders. Among the limiting factors are technical difficulties related to the physiological measurement of human circadian rhythm expression. To better understand human circadian rhythm generation in patients, the implications of circadian rhythm disturbances in pathogenesis, as well as to evaluate potential pharmaceutical treatments, it is important to develop a non-invasive, simple, rapid and reliable in vitro assay to determine circadian rhythm in humans. Recent findings have enabled investigators to demonstrate the feasibility of using peripheral cells to analyze human circadian rhythm. So far, the dermal fibroblast is the only peripheral cell in which stable circadian rhythm can be recorded in humans. However, skin biopsy is not a routine procedure in most clinical departments; they also are painful for patients and often leave scars. Therefore, it would be beneficial to develop an assay system using some other cell type (i.e., a blood cell). In the present proposal, we aim to develop a non-invasive, in vitro assay system to measure human circadian rhythm in macrophages derived from human blood samples, using real-time monitoring of circadian parameters with a clock gene reporter (a total of 31 healthy subjects will be recuited). Once the assay system is established, this technique will be applied to evaluate the effects of chemicals, thereby testing the feasibility of this assay as a future diagnostic tool (a total of 30 paritipants will be recuited). The CRC will be utilized to recruit healthy volunteers and collect blood samples for the above purpose. Each participant will be asked to donate 5 mL blood twice, at least one month apart.