正常光暗周期下大鼠海馬迴中誘導型一氧化氮生成酶之基因表達與機制研究

The Expression and Mechanism of Inducible Nitric Oxide Synthase in Hippocampus of Rats under Normal Light-Dark Cycle

謝家瑜(Chia-Yu Hsieh)

指導教授 ： 王家儀

共同指導教授 ： 李怡萱(Yi-Hsuan Lee)

臺北醫學大學/醫學院/醫學科學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00237

摘要

摘要
「日變節律」是動物經由光照刺激透過視交叉上核，將體內每日的生理週期與外界環境，約略以24小時的節律作同步化規律性循環。研究指出CREB及nNOS在視交叉上核中有節律性表達，而當日變節律受到干擾則會造成iNOS增加，然而在目前仍未知正常光暗周期下iNOS的表達變化。
為了解iNOS在日變節律受到干擾後大量表現的機轉，首先探討iNOS在日變節律中的節律變化，12小時光照-12小時黑暗及24小時全暗的條件下，以RT-PCR探測大鼠大腦皮質、海馬迴及血中iNOS之表達，以及西方墨點分析法觀察海馬迴iNOS及phospho-CREB蛋白質量的變化，並更進一步以染色質免疫沉澱分析法觀察轉錄因子phospho-CREB與iNOS啟動子結合的現象。結果發現海馬迴iNOS基因表達高峰期出現在ZT 6及ZT 22；海馬迴iNOS 蛋白質則呈現節律性的表達，而其高峰期出現在ZT 18出現，且發現iNOS蛋白質量於iNOS mRNA表達增加的時間點-ZT 22之後有逐漸增加的現象，於ZT 8達到高峰；再者調控日變節律基因表達之轉錄因子的實驗結果顯示，磷酸化態的CREB蛋白質量於黑暗期比光亮期多，其高峰則出現在ZT 18及ZT 22，而轉換到光亮期後，CREB磷酸化態蛋白質則呈現低點；而由在染色質免疫沉澱分析
法的實驗中也證實於ZT18，磷酸化態CREB會結合至iNOS啟動子上；再者我們發現血中iNOS基因表達與海馬迴iNOS基因表達呈現同步的現象。
綜合以上之研究顯示海馬迴iNOS在正常光暗周期中存在日變節律，且會透過CREB的磷酸化調節iNOS基因的表達。

關鍵字

光暗周期 ； 海馬迴 ； 誘導型一氧化氮生成酶cAMP response element binding protein (CREB)

並列摘要

Abstract
Suprachiasmatic nucleus (SCN) is the circadian pacemaker of the brain. This clock can be modulated by afferent inputs in response to a variety of external and internal cues. Recent studies showed that cAMP response element binding protein (CREB) and neuronal nitric oxide synthase (nNOS) are rhythmically expressed in SCN. On the other hand, circadian rhythm disturbance induced expression of inducible NOS (iNOS). However, how iNOS expressed rhythmicallly in normal circadian rhythm remains unknown. We explored the circadian rhythm of iNOS expression by RT-PCR in detection of iNOS mRNA in cerebral cortex, hippocampus and peripheral blood；Western blot analysis was used for determination of iNOS protein and phosphorylated CREB under a 12-hour light / dark cycle and 24-hour darkness condition. The chromatin immunoprecipitation (ChIP) assay was used for figuring out the transcriptional event of iNOS expression. The results showed that the rhythms of iNOS mRNA expression had increasing peaks at ZT 6 and ZT 22, and iNOS protein level also had rhythmically expression with peak at ZT 18. Remarkably, iNOS protein levels were increased in light phase after ZT 22 which is the time of the increasing peak of iNOS mRNA. Furthermore, phospho-CREB, the activated form of CREB, had higher expressed levels at ZT 18 and ZT 22, and in ChIP assay showed that phospho-CREB bound to iNOS promoter at ZT 18. Whereas, there had no significant changes on phospho-CREB and iNOS under the 24-hour darkness condition without light-darkness shifting. Interestingly, we found that the level of iNOS mRNA in hippocampus is closely parallel and correlated with the level of iNOS mRNA in the peripheral blood. In conclusion, we demonstrated that iNOS is rhythmically expressed in hippocampus during normal light-dark cycle and it seems to be mediated by the activation of CREB, which might be affected by light-dark cycle.

並列關鍵字

light-dark cycle ； hippocampus ； iNOS ； cAMP response element binding protein (CREB)

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