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Development of culture system of mammalian circadian oscillator and its molecular analysis

哺乳动物昼夜节律振荡器培养体系的研制及其分子分析

基本信息

批准号：
02454105
负责人：
ETOH Teiichi
金额：
$ 3.07万
依托单位：
University of Miyazaki
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

1. Culture of rat suprachiasmatic nucleus neuron: We have developed a dissociated suprachiasmatic nucleus (SCN) cell culture system in order to begin a cellular analysis of mammalian circadian oscillator. Twenty seven hour oscillation of vasopressin release was observed in SCN cell culture. This finding suggest that cultured SCN neurons retain the capacity for circadian oscillation for a long time, and should provide a useful model to analyze the mammalian clock mechanism.2. Reentrainment of circadian clock itself to new light-dark cycle: We investigated whether the circadian clock itself reentrain more quickly to a new light-dark cycle than does the overt rhythm of activity.It took 2.4 days and 6.4 days for clock itself and overt activity rhythm to re-entrain to the new light-dark cycle, respectively. This results suggest that the circadian clock itself reentrain very rapidly following change of light-darkcycle, while the overt rhythms of activity take many cycles.3. Search of proteins related to circadian clock mechanism: We investigated the time-dependent protein in SCN cells harvested every 3 hour from rats kept under constant dark conditions. A 64-kDa protein synthesis rate was rhythmic, and showed two distinct peaks which one of them are observed at around CT 6-9, and the other around CT 21. The peak times consisted with the region of phase-advance induced light and protein synthesis inhibitor.These results indicate the 64kDa protein in SCN may be involved in circadian clock mechanism.4. We examined the difference of circadian clock mechanism between various species of avian, such as chick, crow, pigeon, owl, sparrow, and quail. The pineal gland of owl did not show the circadian rhythm of melatonin release. On the other hand the other avian, except a quail, showed the circadian oscillation of melatonin release under both LD cycle and DD conditions. These results suggest that circadian clock mechanism of avian pineal is different among the species.

1.大鼠视交叉上核神经元的培养：为了开始哺乳动物昼夜节律振荡器的细胞分析，我们建立了一个分离的视交叉上核（SCN）细胞培养系统。在SCN细胞培养中观察到27小时的加压素释放振荡。这一发现表明，培养的SCN神经元在很长一段时间内保持了昼夜节律振荡的能力，为分析哺乳动物的生物钟机制提供了一个有用的模型.生物钟自身对新的光暗周期的再引导：我们研究了生物钟自身是否比外显活动节律更快地再引导到新的光暗周期，生物钟自身和外显活动节律分别需要2.4天和6.4天才能再引导到新的光暗周期。这一结果表明，生物钟本身随着光暗周期的变化而迅速恢复，而活动的外显节律则需要许多周期.与生物钟机制相关的蛋白质的搜索：我们研究了在恒定黑暗条件下每3小时从大鼠收集的SCN细胞中的时间依赖性蛋白质。64-kDa蛋白质合成速率是有节律的，并且显示出两个不同的峰，其中一个在CT 6-9附近观察到，另一个在CT 21附近观察到。峰值出现的时间与相位提前诱导的光和蛋白质合成抑制剂区域一致，表明SCN中的64 kDa蛋白可能参与了生物钟机制.我们研究了不同种类的鸟类，如鸡，乌鸦，鸽子，猫头鹰，麻雀和鹌鹑之间的昼夜节律钟机制的差异。猫头鹰松果体分泌褪黑素的昼夜节律不明显。另一方面，其他鸟类，除了鹌鹑，表现出昼夜振荡的褪黑激素释放的LD周期和DD条件下。这些结果表明鸟类松果体的昼夜节律钟机制在物种之间存在差异。