摘要
昼夜节律生物钟(circadian clock)参与调控机体行为与各项生理功能,以近24小时为周期的节律性震荡来适应不断变化的外界环境。昼夜节律的产生依赖于各种生物钟基因(clock gene)之间的协调表达。哺乳动物的中枢生物钟(central clock)位于下丘脑的视交叉上核(superachiasmatic nuclei, SCN)。外周组织如心、肝、肾等也有生物钟基因的表达。生物钟基因可以通过调节下游的钟控基因(clock controlled genes, ccgs)调节多项生理功能。外周细胞也有生物钟基因的表达,可独立地产生以24小时为周期的生物节律。
研究表明,昼夜节律生物钟不仅参与生理功能的调节,一些生物钟基因表达的改变还参与了心血管系统、肿瘤等疾病的发生发展。在心血管系统疾病中,心肌梗塞(acute myocardial infarction)、脑卒中(stroke)等急性心血管事件的发病具有明显的昼夜节律,通常在清晨高发,但具体机制尚未阐明。本课题组的前期研究发现,apoE-/-小鼠的生物钟基因和凋亡、凝血、血管收缩及脂代谢等生物钟调节基因的表达发生改变。此外,目前生物钟基因与脂质代谢紊乱之间的关系开始受到关注。但新生小鼠最早开始表达生物钟节律的确切时间尚未有相关报道。因此我们首先要明确新生小鼠中枢和心脏生物钟基因节律产生的时间,进一步研究高脂对C57BL/6J小鼠心肌细胞生物钟基因表达的影响,从而探讨生物钟基因与脂质代谢紊乱之间的关系。
第一部分：C57BL/6J小鼠心脏和中枢生物钟基因表达产生昼夜节律的时间
目的：明确小鼠心脏和中枢生物钟基因表达产生昼夜节律的时间。
材料与方法：将C57BL/6J孕鼠在12小时光照/12小时黑暗(Light12h/Dark12h, LD)环境中饲养。乳鼠出生的当天定为P0(postnatal day 0)。在乳鼠出生后的第一天(P1),第三天(P3)和第五天(P5),根据Zeitgaber Time,在ZT0、ZT4、ZT8、ZT12、ZT16、ZT20六个时间点分别处死乳鼠三只(性别不限)。取乳鼠心脏液氮速冻,取脑分离下丘脑视交叉上核(SCN)后液氮速冻,-80℃冰箱保存。利用Real-timeRCR检测C57BL/6J乳鼠SCN和心脏生物钟基因mPer2、mBmal1、mCry1、mRev-erba在不同时间点的表达。
结果：C57BL/6J乳鼠心脏生物钟基因在出生后第三天(P3)表达出昼夜节律,SCN生物钟基因则在第五天(P5)产生了昼夜节律。另外,乳鼠心脏和SCN中的生物钟基因的节律相位会随着出生时间移动,直到第五天(P5)两者产生同步化。
第二部分：高脂对小鼠心肌细胞生物钟基因表达的影响
目的：研究高脂对C57BL/6J小鼠心肌细胞生物钟基因表达的影响。
材料与方法：采用出生三天的C57BL/6J乳鼠,取其心脏培养心肌细胞。48小时后采用血清休克的方法处理心肌细胞,即用含50%马血清的培养液加入细胞两个小时,心肌细胞中的生物钟基因会产生周期大约为24小时的节律；然后在无血清的培养液中加入5种不同浓度(0.5、1、2.5、5、10mmol/L)的甘油三酯,0mmol/L为对照组。根据Zeitgaber Time,加入50%马血清的时间记为ZT0,以后每四个小时提取一次细胞mRNA,,-80℃冰箱保存。用Real-time RCR的方法检测C57BL/6J乳鼠心肌细胞中五个主要的生物钟基因mPer2、mBmal1、mCry1、mRev-erb a和mClock在不同时间点的表达。
结果：研究发现采用血清休克方法处理的心肌细胞内的生物钟基因mRNA从ZT12到ZT60表达了两个完整的昼夜节律周期,且第二个周期的高峰比第一个低。加入甘油三酯后,中浓度组(2.5mmol/L)生物钟基因mRNA表达水平与对照组基本相同；低浓度组(0.5, 1mmol/L)比对照组生物钟基因mRNA表达水平低；高浓度组(5, 10mmol/L)比对照组生物钟基因mRNA表达水平高。
结论：
1、C57BL/6J小鼠心脏和中枢生物钟基因表达昼夜节律的时间分别为出生后第三天和第五天；中枢和外周生物钟基因表达同步化时间为出生后第五天。
2、采用血清休克方法处理的心肌细胞生物钟基因mRNA从ZT12到ZT60表达了两个完整的昼夜节律周期,且第二个周期的高峰比第一个低。
3、高脂可以影响心肌细胞中生物钟基因mRNA的表达,表现为低浓度表达水平降低,高浓度表达水平升高。
The circadian clock is involved in regulating behavior and many physiological functions. The rhythms are entrained to the 24h day by the light-dark (LD) cycle to adapt the natural conditions. Circadian rhythms are generated by the transcription-translation feedback loops of the core circadian genes. In mammals, the master circadian clock is located in suprachiasmatic nucleus (SCN) of the hypothalamus. Apart from SCN, it is shown that peripheral tissues (such as heart, liver, kidney, etc) have circadian oscillators. By regulating the clock controlled genes (CCGs), clock genes regulate many physical functions. Clock genes also can be expressed rhythmically in cultured cells after induced by two-hour serum shock.
Many researches show that the circadian clock plays important roles in several pathological processes such as tumor and cardiovascular diseases besides physiological functions. In cardiovascular disease, the occurrence of stroke and acute myocardial infarction showed a circadian rhythm with the peak in the morning hours. Although the precise mechanism underlying the phenomenon is still not clear, circadian clock may involve in the process. Our previous research demonstrated that the expression amplitude and rhythmicity of all the target clock genes and clock controlled genes which controlled apoptosis, blood clotting, vasoconstriction and lipid metabolism was changed in apoE-/-mice, respectively. Besides, it started to be paid attention to the relationship of the clock genes and metabolic disturbance of lipid. However, no report showed the exact day when the clock genes started to show rhythmicity in mouse. We would ascertain the exact day when the clock genes expressed with circadian rhythms in the mouse SCN and heart, then observed the effect of high fat on the expression of circadian genes in mouse cardiomyocytes and approach the relationship of the clock genes and metabolic disturbance of lipid.
Part I The time when clock genes expressed circadian rhythms in C57BL/6J mouse heart and central tissue
Purpose The aim was to ascertain the exact day when the clock genes expressed with circadian rhythms in mouse heart and central tissue.
Design Female C57BL/6J mice were maintained separately on a 12L:12D light-dark cycle. Day of delivery was designated the postnatal day 0 (PO). For postnatal studies of PI, P3 and P5, infants were kept with their mother through the experiment. According to Zeitgeber time (ZT), three pups of mixed sexes were sampled at different time points including ZTO, ZT4, ZT8, ZT12, ZT16 and ZT20. Hearts were harvested, frozen quickly in liquid nitrogen. Brains were taken away from the skull. And SCN was dissected grossly, quick-frozen in liquid nitrogen and stored at-80℃until RNA isolation. The daily expression of four clock genes mRNA (Bmall, Per2, Cryl and Rev-erb alpha) in mouse SCN and heart was measured at P1, P3 and P5 by real-time PCR.
Results:All the studied mice clock genes began to express in a circadian rhythms manner in heart and SCN at P3 and P5, respectively. Interestingly, the daily rhythmic phase of some clock genes shifted during the postnatal days. Moreover, the expressions of clock genes in heart were not synchronized with those in SCN until at P5.
PartⅡEffects of high fat on the expression of circadian genes in mouse cardiomyocytes
Purpose The goal was to detect the impacts of triglycerides (TGs) on the expression of circadian genes in the cultured mouse cardiomyocytes.
Design Hearts of C57BL/6J mice were harvested at postnatal day 3. Cardiomyocytes were isolated from the hearts. After cultured on plates for 48 hours, the cardiomyocytes were treated with 50%horse serum for 2 hours (serum shock).Clock genes could be expressed rhythmically except mClock. Serial concentration (0.5,1,2.5,5,10mmol/L) of TGs was given to the cardiomyocytes in different petri dishes after serum treatment, and the Ommol/L TGs group was the control. According to Zeitgeber time (ZT), the time when to give the TGs was ZTO. Then the clock genes mRNA expression level was detected by real-time PCR every 4 hours in the next 60 hours and stored at-80℃until RNA isolation. There are 5 core circadian genes in mouse such as mBmall (brain and arylhydrocarbon receptor nuclear translocator-like protein 1), mRev-erba (reverse strand of the c-erba), mPer2 (period 2), mCry (Cryptochrome) and mClock which were measured by real-time PCR.
Results We found that the mRNA expressions in the cells started to show 2 circles of daily rhythms from ZT12 to ZT60 after serum treatment, and the peak of the second circle was lower than the first. The mRNA expressions of circadian genes in cardiomyocytes treated with 2.5mmol/L TGs were equal with TGs-free group. However, the expressions of circadian genes in cardiomyocytes were decreased with lower concentration of TGs (0.5, 1mmol/L), and were dramatically increased with higher level of TGs (5,10mmol/L).
Conclusion
1. In mouse, clock genes began to express in a circadian rhythms manner in heart and SCN at P3 and P5 respectively, and the central clock synchronized the peripheral clock as early as P5.
2. The mRNA expressions in the cells started to show 2 circles of daily rhythms from ZT12 to ZT60 after serum treatment, and the peak of the second circle was lower than the first.
3. The mRNA expression of clock genes could be affected by the TGs and the tendency was bidirectional according to the concentration of TGs.
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