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大鼠视交叉上核与松果体中{sl Clock}基因转录的昼夜节律性及不同光反应性

王国卿, 傅春玲, 李建祥, 杜玉珍, 童建

苏州大学医学院基础医学系生理学教研室.江苏,苏州 215123；苏州大学放射医学与公共卫生学院卫生毒理学教研室.江苏,苏州 215123

摘要

该研究旨在观察和比较视交叉上核(suprachiasmatic nucleus, SCN)与松果体(pineal gland, PG)中{sl Clock}基因内源性昼夜转录变化规律以及光照对其的影响。Sprague--Dawley大鼠在持续黑暗(constant darkness, DD)和12 h光照: 12 h黑暗交替(12 hour--light : 12 hour--dark cycle, LD)光制下分别被饲养8周(n=36)和4周(n=36)后，在一昼夜内每隔4 h采集一组SCN和PG组织(n=6)，提取总RNA，用竞争性定量RT--PCR测定不同昼夜时点(circadian times, CT or zeitgeber times, ZT)各样品中{sl Clock}基因的mRNA相对表达量，通过余弦法和Clock Lab软件获取节律参数，并经振幅检验是否存在昼夜节律性转录变化。结果如下：(1) SCN中{sl Clock}基因mRNA的转录在DD光制下呈现昼低夜高节律性振荡变化({sl P}<0.05)，PG中{sl Clock}基因的转录也显示相似的内源性节律外观，即峰值出现于主观夜晚(SCN为CT15，PG为CT18)，谷值位于主观白天(SCN为CT3，PG为CT6) ({sl P}>0.05)。 (2) LD光制下SCN中{sl Clock}基因的转录也具有昼夜节律性振荡({sl P}<0.05)，但与其DD光制下节律外观相比，呈现反时相节律变化({sl P}<0.05)，且其表达的振幅及峰值的mRNA水平均增加({sl P}<0.05)，而PG中{sl Clock}基因在LD光制下转录的相应节律参数变化却恰恰相反({sl P}<0.05)。 (3) 在LD光制下，光照使PG中{sl Clock}基因转录的节律外观反时相于SCN ({sl P}<0.05)，即在SCN和PG的峰值分别出现于光照期ZT10和黑暗期ZT17，谷值分别位于黑暗期ZT22和光照期ZT5。结果表明，{sl Clock}基因的昼夜转录在SCN和PG中存在同步的内源性节律本质，而光导引在这两个中枢核团调节{sl Clock}基因昼夜节律性转录方面有着不同的作用。

关键词： Clock基因; 昼夜节律; 光照; 视交叉上核; 松果体

Circadian rhythms and different photoresponses of {sl Clock} gene transcription in the rat suprachiasmatic nucleus and pineal gland

Wang Guoqing, Fu Chunling, Li Jianxiang, Du Yuzhen, Tong Jian

Department of Physiology, Medical School,Soochow University.Suzhou 215123,Jiangsu；China

Abstract

The aim of this study was to observe and compare the endogenous circadian rhythm and photoresponse of Clock gene transcription in the suprachiasmatic nucleus (SCN) and pineal gland (PG) of rats. With free access to food and water in special darkrooms, Sprague-Dawley rats were housed under the light regime of constant darkness (DD) for 8 weeks (n=36) or 12 hour-light:12 hour-dark cycle (LD) for 4 weeks (n=36), respectively. Then, their SCN and PG were dissected out every 4 h in a circadian day, 6rats at each time (n=6). All animal treatments and sampling during the dark phases were conducted under red dim light (＜0.1 lux). The total RNA was extracted from each sample and the semi-quantitative RT-PCR was used to determine the temporal mRNA changes of Clock gene in the SCN and PG at different circadian times (CT) or zeitgeber times (ZT). The grayness ratio of Clock/H3.3 bands was served as the relative estimation of Clock gene expression. The experimental data were analyzed by the Cosine method and the Clock Lab software to fit original results measured at 6 time points and to simulate a circadian rhythmic curve which was then examined for statistical difference by the amplitude F test. The main results are as follows: (1) The mRNA levels of Clock gene in the SCN under DD regime displayed the circadian oscillation (P＜0.05). The endogenous rhythmic profiles of Clock gene transcription in the PG were similar to those in the SCN (P＞0.05) throughout the day with the peak at the subjective night (CT15 in the SCN or CT18 in the PG)and the trough during the subjective day (CT3 in the SCN or CT6 in the PG). (2) Clock gene transcription in the SCN under LD cycle also showed the circadian oscillation (P＜0.05), and the rhythmic profile was anti-phasic to that under DD condition (P＜0.05). The amplitude and the mRNA level at the peak of Clock gene transcription in the SCN under LD were significantly increased compared with that under DD (P＜0.05), while the value of corresponding rhythmic parameters in the PG under LD were remarkably decreased (P＜0.05).(3) Under LD cycle, the circadian profiles of Clock gene transcription induced by light in the PG were quite different from those in the SCN (P＜0.05). Their Clock transcription rhythms were anti-phasic, i.e., showing peaks at the light phase ZT10 in the SCN or at the dark time ZT17 in the PG and troughs during the dark time ZT22 in the SCN or during the light phase ZT5 in the PG. The findings of the present study indicate a synchronous endogenous nature of the Clock gene circadian transcriptions in the SCN and PG, and different roles of light regime in modulating the circadian transcriptions of Clock gene in these two central nuclei.

Key words: Clock gene;Circadian rhythm;Light;Suprachiasmatic nucleus;pineal gland

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引用本文：

王国卿, 傅春玲, 李建祥, 杜玉珍, 童建. 大鼠视交叉上核与松果体中{sl Clock}基因转录的昼夜节律性及不同光反应性[J]. 生理学报 2006; 58 (4): .

Wang Guoqing, Fu Chunling, Li Jianxiang, Du Yuzhen, Tong Jian. Circadian rhythms and different photoresponses of {sl Clock} gene transcription in the rat suprachiasmatic nucleus and pineal gland. Acta Physiol Sin 2006; 58 (4): (in Chinese with English abstract).