發(fā)布時間：2018-07-12 12:46

  本文選題：生物鐘基因 + 小鼠　； 參考：《復(fù)旦大學(xué)》2010年碩士論文

【摘要】： 晝夜節(jié)律生物鐘(circadian clock)參與調(diào)控機體行為與各項生理功能,以近24小時為周期的節(jié)律性震蕩來適應(yīng)不斷變化的外界環(huán)境。晝夜節(jié)律的產(chǎn)生依賴于各種生物鐘基因(clock gene)之間的協(xié)調(diào)表達。哺乳動物的中樞生物鐘(central clock)位于下丘腦的視交叉上核(superachiasmatic nuclei, SCN)。外周組織如心、肝、腎等也有生物鐘基因的表達。生物鐘基因可以通過調(diào)節(jié)下游的鐘控基因(clock controlled genes, ccgs)調(diào)節(jié)多項生理功能。外周細胞也有生物鐘基因的表達,可獨立地產(chǎn)生以24小時為周期的生物節(jié)律。 研究表明,晝夜節(jié)律生物鐘不僅參與生理功能的調(diào)節(jié),一些生物鐘基因表達的改變還參與了心血管系統(tǒng)、腫瘤等疾病的發(fā)生發(fā)展。在心血管系統(tǒng)疾病中,心肌梗塞(acute myocardial infarction)、腦卒中(stroke)等急性心血管事件的發(fā)病具有明顯的晝夜節(jié)律,通常在清晨高發(fā),但具體機制尚未闡明。本課題組的前期研究發(fā)現(xiàn),apoE-/-小鼠的生物鐘基因和凋亡、凝血、血管收縮及脂代謝等生物鐘調(diào)節(jié)基因的表達發(fā)生改變。此外,目前生物鐘基因與脂質(zhì)代謝紊亂之間的關(guān)系開始受到關(guān)注。但新生小鼠最早開始表達生物鐘節(jié)律的確切時間尚未有相關(guān)報道。因此我們首先要明確新生小鼠中樞和心臟生物鐘基因節(jié)律產(chǎn)生的時間,進一步研究高脂對C57BL/6J小鼠心肌細胞生物鐘基因表達的影響,從而探討生物鐘基因與脂質(zhì)代謝紊亂之間的關(guān)系。 第一部分：C57BL/6J小鼠心臟和中樞生物鐘基因表達產(chǎn)生晝夜節(jié)律的時間 目的：明確小鼠心臟和中樞生物鐘基因表達產(chǎn)生晝夜節(jié)律的時間。 材料與方法：將C57BL/6J孕鼠在12小時光照/12小時黑暗(Light12h/Dark12h, LD)環(huán)境中飼養(yǎng)。乳鼠出生的當(dāng)天定為P0(postnatal day 0)。在乳鼠出生后的第一天(P1),第三天(P3)和第五天(P5),根據(jù)Zeitgaber Time,在ZT0、ZT4、ZT8、ZT12、ZT16、ZT20六個時間點分別處死乳鼠三只(性別不限)。取乳鼠心臟液氮速凍,取腦分離下丘腦視交叉上核(SCN)后液氮速凍,-80℃冰箱保存。利用Real-timeRCR檢測C57BL/6J乳鼠SCN和心臟生物鐘基因mPer2、mBmal1、mCry1、mRev-erba在不同時間點的表達。 結(jié)果：C57BL/6J乳鼠心臟生物鐘基因在出生后第三天(P3)表達出晝夜節(jié)律,SCN生物鐘基因則在第五天(P5)產(chǎn)生了晝夜節(jié)律。另外,乳鼠心臟和SCN中的生物鐘基因的節(jié)律相位會隨著出生時間移動,直到第五天(P5)兩者產(chǎn)生同步化。 第二部分：高脂對小鼠心肌細胞生物鐘基因表達的影響 目的：研究高脂對C57BL/6J小鼠心肌細胞生物鐘基因表達的影響。 材料與方法：采用出生三天的C57BL/6J乳鼠,取其心臟培養(yǎng)心肌細胞。48小時后采用血清休克的方法處理心肌細胞,即用含50%馬血清的培養(yǎng)液加入細胞兩個小時,心肌細胞中的生物鐘基因會產(chǎn)生周期大約為24小時的節(jié)律；然后在無血清的培養(yǎng)液中加入5種不同濃度(0.5、1、2.5、5、10mmol/L)的甘油三酯,0mmol/L為對照組。根據(jù)Zeitgaber Time,加入50%馬血清的時間記為ZT0,以后每四個小時提取一次細胞mRNA,,-80℃冰箱保存。用Real-time RCR的方法檢測C57BL/6J乳鼠心肌細胞中五個主要的生物鐘基因mPer2、mBmal1、mCry1、mRev-erb a和mClock在不同時間點的表達。 結(jié)果：研究發(fā)現(xiàn)采用血清休克方法處理的心肌細胞內(nèi)的生物鐘基因mRNA從ZT12到ZT60表達了兩個完整的晝夜節(jié)律周期,且第二個周期的高峰比第一個低。加入甘油三酯后,中濃度組(2.5mmol/L)生物鐘基因mRNA表達水平與對照組基本相同；低濃度組(0.5, 1mmol/L)比對照組生物鐘基因mRNA表達水平低；高濃度組(5, 10mmol/L)比對照組生物鐘基因mRNA表達水平高。 結(jié)論： 1、C57BL/6J小鼠心臟和中樞生物鐘基因表達晝夜節(jié)律的時間分別為出生后第三天和第五天；中樞和外周生物鐘基因表達同步化時間為出生后第五天。 2、采用血清休克方法處理的心肌細胞生物鐘基因mRNA從ZT12到ZT60表達了兩個完整的晝夜節(jié)律周期,且第二個周期的高峰比第一個低。 3、高脂可以影響心肌細胞中生物鐘基因mRNA的表達,表現(xiàn)為低濃度表達水平降低,高濃度表達水平升高。
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363

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本文編號：2117203