高脂对小鼠心肌细胞生物钟基因表达的影响

发布时间：2018-07-12 12:46

本文选题：生物钟基因 + 小鼠　；参考：《复旦大学》2010年硕士论文

【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。
[Abstract]:The circadian rhythmic biological clock (circadian clock) participates in the regulation of the body's behavior and various physiological functions. The circadian rhythmic oscillation is adapted to the changing external environment for nearly 24 hours. The occurrence of the circadian rhythm depends on the coordination table between the various biological clock genes (clock gene). The central biological clock (central clock) of the mammal is located at the center of the circadian clock. The suprachiasmatic nucleus of the hypothalamus (superachiasmatic nuclei, SCN). Peripheral tissues such as the heart, liver and kidney also have the expression of a biological clock gene. The clock gene can regulate a number of physiological functions by regulating the clock controlled genes (CCGs) downstream. The peripheral cells also have the expression of the clock gene of the biological clock, which can be produced independently for 24 hours. A cycle of biological rhythms.
The circadian clock not only participates in the regulation of physiological function, but also changes in the gene expression of some biological clocks in the cardiovascular system, tumor and other diseases. In cardiovascular system diseases, acute cardiovascular events such as myocardial infarction (acute myocardial infarction), and cerebral apoplexy (stroke) are obvious. The circadian rhythm is usually high in the early morning, but the specific mechanism has not been clarified. Previous studies in this group have found that the expression of clock gene and apoptosis, blood clotting, vasoconstriction and lipid metabolism in apoE-/- mice changed. In addition, the relationship between biological Zhong Jiyin and lipid metabolism began to be concerned. However, the exact time that the newborn mice first began to express the circadian rhythm of the biological clock has not yet been reported. Therefore, we should first clarify the time of the generation of the gene rhythm of the central and cardiac circadian clock in newborn mice, and further study the effect of high fat on the gene expression of the clocks of C57BL/6J mice, thus exploring the biological clock gene and lipid metabolism. The relationship between disorder.
Part one: the circadian rhythm of gene expression in cardiac and central circadian rhythms of C57BL/6J mice
Objective: to determine the circadian rhythm of gene expression in cardiac and central circadian mice.
Materials and methods: C57BL/6J pregnant rats were raised in the /12 hour dark (Light12h/Dark12h, LD) environment at 12 small hours. The day of birth was P0 (postnatal day 0). The first day after birth (P1), third days (P3) and fifth days (P5) after the birth of the milk rat, the milk was executed at six times according to Zeitgaber Time. Three rats (sex unlimited). Take liquid nitrogen quick freeze in the heart of the rat, take the cerebral separation of the hypothalamic suprachiasmatic nucleus (SCN) and keep the liquid nitrogen quick freezing, and keep the refrigerator at -80 C. The expression of SCN and cardiac clock gene mPer2, mBmal1, mCry1 and mRev-erba at different time points are detected by Real-timeRCR.
Results: the circadian rhythm of the cardiac clock gene in C57BL/6J rats was expressed at third days after birth (P3), and the circadian rhythm of the SCN clock gene was fifth days (P5). In addition, the rhythmic phase of the clock genes in the heart and SCN of the milk mice moved with the time of birth, until the fifth days (P5) were synchronized.
The second part: the effect of high fat on the expression of circadian clock genes in mouse cardiomyocytes.
Objective: To study the effects of high fat on the expression of circadian clock genes in cardiac myocytes of C57BL/6J mice.
Materials and methods: the cardiac myocytes were treated with the method of serum shock after.48 hours of heart culture of C57BL/6J milk mice born for three days. That is to say, the cells with 50% horse serum were added to the cell for two hours. The circadian clock gene in the cardiac myocytes produced about 24 hours of rhythm; then it was serum-free. In the medium, 5 kinds of triglycerides (0.5,1,2.5,5,10mmol/L) and 0mmol/L were added to the control group. According to Zeitgaber Time, the time of adding 50% horse serum was recorded as ZT0, mRNA was extracted once every four hours, and -80 centigrade refrigerator was preserved. The five main biological clocks in the cardiomyocytes of C57BL/6J milk mice were detected by Real-time RCR. Expression of genes mPer2, mBmal1, mCry1, mRev-erb A and mClock at different time points.
Results: the study found that the biological clock gene mRNA in the cardiac myocytes treated with serum shock method expressed two complete circadian rhythm cycles from ZT12 to ZT60, and the peak of the second cycles was lower than that of the first. After adding triglycerides, the expression level of mRNA in the medium concentration group (2.5mmol/L) was the same as that of the control group; The concentration group (0.5, 1mmol/L) was lower than that of the control group, and the expression level of mRNA was lower than that of the control group. The expression of mRNA was higher in the high concentration group (5, 10mmol/L) than in the control group.
Conclusion:
1, the day and night rhythm of the cardiac and central clock genes in C57BL/6J mice were third and fifth days after birth, and the synchronization time of gene expression in the central and peripheral circadian clock was fifth days after birth.
2, the cardiac cell clock gene mRNA, treated with a serum shock method, expressed two complete circadian rhythmic cycles from ZT12 to ZT60, and the peak of the second cycles was lower than that of the first.
3, high fat can affect the expression of mRNA in cardiac myocytes, showing a low level of expression and a high level of expression.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【共引文献】