本文選題：單色光 + 鐘基因　； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：禽類褪黑激素的分泌呈現(xiàn)晝低夜高的節(jié)律性。該節(jié)律性分泌受到體內(nèi)生物鐘系統(tǒng)的調(diào)控。松果體和下丘腦均屬于禽類生物鐘的主鐘振蕩器,二者既可以獨(dú)立振蕩又能夠相互作用。生物鐘振蕩器的振蕩依賴于一個(gè)由正調(diào)控鐘基因(cClock、cBmalll及cBmall2)和負(fù)調(diào)控鐘基因(cCry1、cCry2、cPer2及cPer3)組成的負(fù)反饋調(diào)節(jié)環(huán)路。前期研究顯示單色綠光可以提高雛雞血漿褪黑激素水平,但該作用的機(jī)制是否是通過影響雞生物鐘系統(tǒng)而進(jìn)行調(diào)節(jié)的尚不清楚。因此,本研究選用剛出殼的AA肉雞分別飼養(yǎng)于白(400-760nm)、紅(660nm)、綠(560nm)、藍(lán)(480nm)四種光色下,光照制度LD 12:12,并于第三日齡實(shí)施松果體摘除手術(shù)。飼養(yǎng)至第14日齡,每隔4小時(shí),共6個(gè)時(shí)間點(diǎn)取松果體和下丘腦,研究不同單色光對(duì)雞松果體和下丘腦生物鐘晝夜節(jié)律系統(tǒng)的影響,探討單色光影響松果體褪黑激素分泌的機(jī)制以及松果體褪黑激素在單色光對(duì)下丘腦生物鐘晝夜振蕩器影響中的作用。研究結(jié)果如下:1雞松果體和下丘腦鐘基因的晝夜節(jié)律表達(dá)雞松果體和下丘腦正、負(fù)調(diào)控鐘基因均表現(xiàn)出明顯的晝夜節(jié)律性表達(dá)。除了cP rs基因,其他鐘基因的表達(dá)均表現(xiàn)為白天升高、夜間降低的形式。在兩個(gè)主鐘中,cCrys和cPers鐘基因表達(dá)相似,峰相位較為接近;但下丘腦cClock的振幅是松果體的兩倍,而松果體cBBmals和cCry1的振幅約為下丘腦的兩倍,松果體cClock、cBmas和cPer3的峰相位較下丘腦的延遲。2單色光對(duì)松果體生物鐘晝夜系統(tǒng)的影響對(duì)鐘基因晝夜表達(dá)的影響:不同單色光照射下,松果體鐘基因依然維持明顯的晝夜節(jié)律性表達(dá),除了紅光下cCry2的節(jié)律性較白光下明顯降低(R2 = 0.243,P = 0.033)。綠光明顯增加正調(diào)控鐘基因的表達(dá)量,降低負(fù)調(diào)控鐘基因的表達(dá)量;而紅光則抑制了正調(diào)控鐘基因的表達(dá)量,增加了負(fù)調(diào)控鐘基因的表達(dá)量。與白光相比,綠光下正調(diào)控基因的中值和振幅均增加,負(fù)調(diào)控基因的中值和振幅則減小。在藍(lán)光影響下,正調(diào)控鐘基因的相位均較白光下發(fā)生延遲(0.2 h-0.8 h),負(fù)調(diào)控鐘基因的相位均發(fā)生提前(0.1 h-1.5 h)。同時(shí),綠光也明顯增加了鐘蛋白CLOCK和BMAL1的表達(dá)量。對(duì)褪黑激素晝夜含量的影響:綠光可以顯著提高松果體cAana如基因表達(dá)的水平,同時(shí)綠光下血漿褪黑激素的含量也高出其他光組1.48%-30.93%(P = 0.000-0.655,ANOVA);而紅光下褪黑激素的含量明顯較其他光組低了 5.6%-23.4%(P=0.000-0.006,ANOVA)。不同光色下松果體cAanat和血漿褪黑激素均表現(xiàn)出明顯的晝夜節(jié)律。同時(shí),綠光下松果體cAanat和血漿褪黑激素晝夜振蕩的中值和振幅均高于其他光色,而在紅光下則均低于其他光組。對(duì)松果體視蛋白表達(dá)的影響:白光和綠光下,cOpnp的基因表達(dá)呈現(xiàn)晝高夜低的表達(dá)形式;而藍(lán)光和紅光下,cOpnp則呈現(xiàn)出白天低而夜間高的形式。紅光下cOpnp的振幅明顯下降且晝夜節(jié)律性消失(R2= 0.2,P = 0.425)。cOpn4-1基因的表達(dá)量呈現(xiàn)白天低夜間高的形式。比起其他光色,綠光下cOpn4-1的晝夜節(jié)律性和振幅明顯增加。不同單色光下cOpn4-2均呈現(xiàn)白天高夜間低的晝夜節(jié)律性表達(dá),其中藍(lán)光下cOpn4-2節(jié)律性以及振幅明顯降低。在單色光下cCreb的振蕩均表現(xiàn)出明顯的晝夜節(jié)律性。3單色光對(duì)下丘腦鐘基因晝夜表達(dá)的影響不同光色下,下丘腦鐘基因均呈現(xiàn)明顯的晝夜節(jié)律性表達(dá)。綠光顯著增加了鐘基因cClodk、cBmal1以及cCry1的表達(dá)量,抑制了cBmal 和cCry2的表達(dá);紅光則降低了 cClock、cBmal1以及cCOy1的表達(dá)量,卻明顯增加了 cBBmal2和cCry2的表達(dá)量。就節(jié)律參數(shù)而言,cClock、cBmaal1以及cCry1的中值和振幅在綠光下最大,而cBmal2的中值和振幅在綠光下最小;在紅光,cClock和cBmal1的中值和振幅均較白光和綠光小。同時(shí),紅光下鐘基因峰相位的移動(dòng)比綠光和藍(lán)光更為明顯。對(duì)鐘蛋白的檢測(cè)發(fā)現(xiàn),綠光下CLOCK和BMAL1表達(dá)的變化與基因水平相類似。同時(shí),鐘蛋白CLOCK在下丘腦視交叉上核、前內(nèi)側(cè)核、室周核、室旁核及正中隆起中均有陽性表達(dá)。4松果體褪黑激素介導(dǎo)單色光對(duì)下丘腦鐘基因表達(dá)的影響松果體摘除對(duì)血漿褪黑激素晝夜含量的影響:松摘后不同光色下雞血漿褪黑激素水平與各自假手術(shù)組相比均明顯下降,但依然保持明顯的晝夜節(jié)律性;同時(shí)綠光下血漿褪黑激素水平仍然高出其他光色 7.0%-55.52%(P= 0.000-0.039,ANOVA)。松果體摘除對(duì)下丘腦鐘基因表達(dá)的影響:隨著松摘后褪黑激素水平的下降,在不同光色下下丘鐘基因cClock和cPer2的振幅均較各自假手術(shù)組明顯下降;而cBmal1的中值和振幅較假手術(shù)組相似,但紅光下和藍(lán)光下的相位分別較各自假手術(shù)組延遲2 h和提前2 h。紅光下下丘腦cBmal2和cCry2的晝夜表達(dá)均失去了節(jié)律性。松摘后綠光下cCry1的表達(dá)量下降,而紅光下表達(dá)量升高。松摘后紅光下cPer3的表達(dá)量依然高于其他光組。同時(shí),下丘腦鐘蛋白CLOCK的定位仍然出現(xiàn)在下丘腦視覺視交叉上核、室周核以及室旁核。松果體摘除對(duì)下丘腦褪黑激素受體表達(dá)的影響:14日齡雞下丘腦中褪黑激素受體cMel1a、cMel1b和cMel1c均呈現(xiàn)明顯的晝夜節(jié)律性表達(dá)。松摘后三種受體的表達(dá)量較對(duì)照組和假手術(shù)組均分別顯著降低 35.43%-37.98%(P= 0.000,AVONA)、12.55%-34.55%(P=0.000-0.032,AVONA)和4.39%-23.71%(P = 0.000-0.999,AVONA)。同時(shí),松摘后三種受體的振幅較各自對(duì)照組和假手術(shù)組明顯下降;cMel1b和cMel1c表達(dá)的晝夜節(jié)律性也隨之消失。影響鐘基因表達(dá)的褪黑激素受體途徑:體外添加褪黑激素濃度250 pg/mL時(shí),鐘基因cClock的表達(dá)量顯著高于未添加褪黑激素組45.63%-45.81%(P = 0.003-0.606,ANOVA)。當(dāng)加入Mella/Mellb非特異性阻斷劑Luzindole時(shí),鐘基因cClock:的表達(dá)量顯著下降30.6%-31.4%(P=0.004-0.01,ANOVA);而在僅加入Mellb特異性受體阻斷劑4P-PDOT的處理組中,鐘基因cClock的表達(dá)量比僅添加褪黑激素刺激組的表達(dá)量顯著低23.6%(P = 0.009,ANOVA);而當(dāng)僅添加褪黑激素受體Mellc特異性受體阻斷劑Prazosin時(shí),鐘基因cC/ock的表達(dá)量高于其他未加褪黑激素組 44.37%-47.6%(P= 0.002-0.009,ANOVA)。結(jié)論:七種核心鐘基因在14日齡雞松果體和下丘腦中的表達(dá)均存在明顯的晝夜節(jié)律性。綠光可以通過影響cOpnp和cOpn4-1來增加松果體正調(diào)控鐘基因的表達(dá)量并降低了負(fù)調(diào)控鐘基因的表達(dá),促進(jìn)松果體褪黑激素關(guān)鍵合成酶cAanat的表達(dá),從而增加血漿褪黑激素水平。同時(shí),松果體摘除前后下丘腦鐘蛋白CLOCK均出現(xiàn)在視覺視交叉上核、室旁核及室周核,單色光可以通過影響松果體褪黑激素的分泌,后者通過其受體Mella和Mellb調(diào)節(jié)下丘腦鐘基因的晝夜節(jié)律表達(dá)。
[Abstract]:The secretion of fowl melatonin appears to be rhythmical at low day night height. The rhythmic secretion is regulated by the body clock system in the body. The pineal and hypothalamus all belong to the main clock oscillator of the bird clock. The two can both oscillate and interact. The oscillation of the biologic oscillator depends on a positive regulatory clock gene (cClock, cB Malll and cBmall2) and negative regulatory clock genes (cCry1, cCry2, cPer2 and cPer3) are a negative feedback regulation loop. Previous studies have shown that monochromatic green light can improve the level of plasma melatonin in chicks, but the mechanism of this effect is not clear by affecting the chicken clock system. Therefore, this study selects the AA meat from the rigid shell. Chickens were fed in four kinds of light colors, white (400-760nm), red (660nm), green (560nm) and blue (480nm). The light system was LD at 12:12, and the pineal fruit was removed at the age of third days. The pineal and hypothalamus were taken at fourteenth days of age and every 4 hours. The circadian rhythms of the pineal and hypothalamus circadian circadian rhythms of different monochromatic light were studied. The effect of monochromatic light on the secretion of melatonin in pineal pineal body and the effect of pineal melatonin on the circadian oscillator of the hypothalamus circadian clock were investigated. The results are as follows: 1 the circadian rhythm of the pineal and hypothalamic clock genes expressed in the pineal and hypothalamus of the chicken and the negative regulatory clock genes were all obvious. The expression of diurnal rhythmic expression. Except cP RS gene, the expression of other clock genes expressed in the form of daytime increase and nocturnal decrease. In the two main clocks, the expression of cCrys and cPers clock genes is similar, the peak phase is close, but the amplitude of the cClock in the hypothalamus is two times that of the pineal body, and the amplitude of the pineal cBBmals and cCry1 is about two times that of the hypothalamus. The peak phase of the pineal cClock, cBmas and cPer3 is compared to the effect of the delayed.2 monochromatic light on the circadian clock gene expression in the pineal body clock in the hypothalamus. Under different monochromatic light, the pineal clock gene still maintains an obvious circadian expression, but the rhythm of cCry2 is significantly lower than the white light under the red light (R2 = 0). .243, P = 0.033). The green light obviously increased the expression of the positive clock gene and reduced the expression of the negative regulation clock gene, while red light inhibited the expression of the positive clock gene and increased the expression of the negative regulation clock gene. Compared with the white light, the median and amplitude of the positive regulation genes under the green light were increased, the median and amplitude of the negative regulation genes were increased. The phase of the positive clock gene was delayed (0.2 h-0.8 h) under the influence of blue light (0.2 h), and the phase of the negative regulatory clock gene was advanced (0.1 h-1.5 h). Meanwhile, green light also significantly increased the expression of the clock protein CLOCK and BMAL1. At the same time, the content of melatonin in the green light was also higher than that of other light groups 1.48%-30.93% (P = 0.000-0.655, ANOVA), while the content of melatonin under red light was significantly lower than that of other light groups (P=0.000-0.006, ANOVA). The circadian rhythm of the pineal cAanat and plasma melatonin in different light colors showed obvious circadian rhythm. The median and amplitude of the diurnal oscillations of the pineal pineal body cAanat and the plasma melatonin were higher than those of other light colors, but under the red light, the effects on the expression of pineal stereopacs: white light and green light, the expression of gene expression of cOpnp presented a high day low expression form, while under blue and red light, cOpnp showed low daytime. In the form of high night, the amplitude of cOpnp decreased obviously under the red light and the circadian rhythm disappeared (R2= 0.2, P = 0.425), the expression of.COpn4-1 gene showed low night high in the daytime. Compared with the other light colors, the circadian rhythm and amplitude of cOpn4-1 increased obviously under the green light. The cOpn4-2 of different monochromatic light showed high night day night day high day. Rhythmic expression, in which the cOpn4-2 rhythms and the amplitude of the cCreb under blue light were significantly reduced. In the monochromatic light, the circadian rhythm of the circadian.3 monochromatic light affects the diurnal expression of the clock gene in the hypothalamus. The circadian rhythm of the clock genes in the hypothalamus was obviously expressed in the circadian rhythm. The green light significantly increased the clock gene cClodk, C The expression of Bmal1 and cCry1 inhibited the expression of cBmal and cCry2; red light reduced the expression of cClock, cBmal1 and cCOy1, but significantly increased the expression of cBBmal2 and cCry2. In terms of the rhythmic parameters, the median and amplitude of cClock, cBmaal1 and cCry1 were the largest under the green light, and the minimum of the median and amplitude in the green light. The median and amplitude of the red light, cClock and cBmal1 were smaller than the white and green light. At the same time, the phase of the bell gene peak movement was more obvious than the green and blue light. The detection of the clock protein showed that the changes in the expression of CLOCK and BMAL1 under green light were similar to those of the gene level. At the same time, the clock protein CLOCK was in the hypothalamus suprachiasmatic nucleus, the medial nucleus, and the peritrom. The effects of.4 pineal melatonin on the expression of Zhong Jiyin in the hypothalamus were positively expressed in the nucleus, paraventricular nucleus and median eminence. The effects of pineal extirpation on the day and night content of melatonin in the pineal body: the levels of melatonin in the plasma of chickens were significantly lower than those in the sham groups after the release. The plasma melatonin level under the green light was still higher than the other light color 7.0%-55.52% (P= 0.000-0.039, ANOVA). The effect of pineal extirpation on the expression of the hypothalamus clock gene: the amplitude of cClock and cPer2 in the hypothalamus gene cClock and cPer2 in different light colors were significantly lower than those of the respective sham groups. The median and amplitude of cBmal1 were similar to those of the sham operation group, but the phase of the under and blue light under the red light and the blue light lost the rhythm of the diurnal expression of cBmal2 and cCry2 in the hypothalamus under the red light of 2 h. and the delay of 2 h in the sham operation group and the red light under the red light. The expression of the hypothalamic clock protein CLOCK still appears in the hypothalamic visual actin supraventricular nucleus, perinuclear nucleus and paraventricular nucleus. The effect of pineal extirpation on the expression of melatonin receptor in the hypothalamus: melatonin receptor cMel1a, cMel1b, and cMel1c in the hypothalamus of 14 day old chickens all present a significant diurnal node The expression of three receptors decreased 35.43%-37.98% (P= 0, AVONA), 12.55%-34.55% (P=0.000-0.032, AVONA) and 4.39%-23.71% (P = 0.000-0.999, AVONA), respectively. Meanwhile, the amplitude of the three receptors was significantly lower than that of the control group and the sham operation group; cMel1b and cMel1c tables were significantly decreased. The circadian rhythm of the clock was also disappearing. The expression of melatonin receptor in the clock gene expression: when the concentration of melatonin was 250 pg/mL, the expression of clock gene cClock was significantly higher than that of 45.63%-45.81% without melatonin group (P = 0.003-0.606, ANOVA). When Mella/ Mellb non-specific blocker Luzindole was added, the clock gene cClock: was in cClock:. The expression was significantly decreased by 30.6%-31.4% (P=0.004-0.01, ANOVA), and the expression of clock gene cClock was significantly lower than that of only melatonin stimulant group (P = 0.009, ANOVA) in the treatment group only adding Mellb specific receptor blocker 4P-PDOT, while only melatonin receptor Mellc specific receptor blocker Prazosin was added to 30.6%-31.4%. The expression of clock gene cC/ock was higher than that of other non melatonin group 44.37%-47.6% (P= 0.002-0.009, ANOVA). Conclusion: the expression of the seven core clock genes in the pineal body and the hypothalamus of 14 days of age had obvious circadian rhythms. Green light could increase the expression of the pineal clock gene by affecting the cOpnp and cOpn4-1. The expression of the pineal melatonin key synthetase cAanat was reduced and the plasma melatonin level was increased. At the same time, the hypothalamic clock protein CLOCK appeared in the supraventricular nucleus, paraventricular nucleus and perineal nucleus before and after the removal of pineal body, and monochromatic light could affect the secretion of melatonin in the pineal body. The latter regulates circadian rhythm of the hypothalamic clock gene through its receptors Mella and Mellb.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S831

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