IN VITRO DIAGNOSTIC TEST FOR LITHIUM-SENSITIVE BIPOLAR DISORDER

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。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 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的研究机构。 我们目前的建议的一个目标是建立一个测定系统，通过该系统可以使用具有昼夜节律报告基因和真实的时间生物发光监测的人成纤维细胞来监测昼夜节律，并测试我们的假设，即可以使用人外周细胞来预测对锂的临床反应。 B.研究和结果。 第一个目标是应用体外昼夜节律记录方法，使用人原代成纤维细胞。到目前为止，招募了11名健康参与者，并建立了6个成纤维细胞系。在培养和扩增后，将生物钟Bmal 1启动子-荧光素酶基因递送至成纤维细胞，并建立Bmal 1-荧光素酶细胞系。我们优化了培养和记录条件。现在已经在这些人成纤维细胞系中记录了稳健和持续的昼夜节律。平均周期长度为24.60 ± 0.16 h（N=5）。该结果与Brown博士及其同事先前发表的结果一致（Brown et al.，2005年，我们完成了目标1。 在目标2中，我们计划检查锂对从锂应答者和无应答者获得的成纤维细胞中的昼夜节律参数的影响。我们一直在优化锂治疗条件，以应用于双相情感障碍患者的细胞。由于在先前的研究中使用高锂浓度来测试锂对周期长度的影响，因此我们测试了3、5、7和10 mM锂刺激对周期长度的影响。同时，我们希望测试锂在治疗浓度下的作用，例如，由于急性1 mM锂刺激没有显示出显著的影响，我们在节律记录之前在1 mM锂的存在下培养细胞一周或四周。我们也选择了四周，因为临床锂敏感性首先在锂治疗后四周确定。

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. A goal of our present proposal is to establish an assay system by which circadian rhythms can be monitored using human fibroblasts with a circadian reporter and real time bioluminescence monitoring, and to test our hypothesis that clinical response to lithium can be predicted using human peripheral cells. b. Studies and Results. The goal of the first Aim is to apply in vitro circadian rhythm recording method using human primary fibroblasts. So far eleven healthy participants were recruited and six fibroblast cell lines have been established. After cultivation and expansion, the circadian clock Bmal1 promoter-luciferase gene was delivered to fibroblasts, and Bmal1-luciferase cell lines were established. We optimized culture and recording conditions. Robust and sustained circadian rhythms have now been recorded in these human fibroblast cell lines. Average period length was 24.60 ¿ 0.16 h (N=5). The results were in agreement with those previously published by Dr. Brown and his colleagues (Brown et al., 2005) and, therefore, we have completed the Aim 1. In Aim 2 we plan to examine the effects of lithium on circadian rhythm parameters in fibroblasts obtained from lithium responders and non-responders. We have been optimizing lithium treatment conditions to be applied to bipolar disorder patients' cells. Since high lithium concentrations were used to test the effects of lithium on the period length in previous studies, we tested 3, 5, 7, and 10 mM lithium stimulation on the period length. At the same time, we want to test the effects of lithium at therapeutic concentrations, e.g., 1 mM. Since acute 1mM lithium stimulation did not show significant impact, we have been culturing cells in the presence of l mM lithium for one or four weeks before rhythm recording. We chose four weeks as well because the clinical lithium sensitivity is first determined four weeks after lithium therapy.