单色光对鸡松果体和下丘脑生物钟昼夜节律系统的影响

发布时间：2018-04-27 06:29

本文选题：单色光 + 钟基因　；参考：《中国农业大学》2017年博士论文

【摘要】：禽类褪黑激素的分泌呈现昼低夜高的节律性。该节律性分泌受到体内生物钟系统的调控。松果体和下丘脑均属于禽类生物钟的主钟振荡器,二者既可以独立振荡又能够相互作用。生物钟振荡器的振荡依赖于一个由正调控钟基因(cClock、cBmalll及cBmall2)和负调控钟基因(cCry1、cCry2、cPer2及cPer3)组成的负反馈调节环路。前期研究显示单色绿光可以提高雏鸡血浆褪黑激素水平,但该作用的机制是否是通过影响鸡生物钟系统而进行调节的尚不清楚。因此,本研究选用刚出壳的AA肉鸡分别饲养于白(400-760nm)、红(660nm)、绿(560nm)、蓝(480nm)四种光色下,光照制度LD 12:12,并于第三日龄实施松果体摘除手术。饲养至第14日龄,每隔4小时,共6个时间点取松果体和下丘脑,研究不同单色光对鸡松果体和下丘脑生物钟昼夜节律系统的影响,探讨单色光影响松果体褪黑激素分泌的机制以及松果体褪黑激素在单色光对下丘脑生物钟昼夜振荡器影响中的作用。研究结果如下:1鸡松果体和下丘脑钟基因的昼夜节律表达鸡松果体和下丘脑正、负调控钟基因均表现出明显的昼夜节律性表达。除了cP rs基因,其他钟基因的表达均表现为白天升高、夜间降低的形式。在两个主钟中,cCrys和cPers钟基因表达相似,峰相位较为接近;但下丘脑cClock的振幅是松果体的两倍,而松果体cBBmals和cCry1的振幅约为下丘脑的两倍,松果体cClock、cBmas和cPer3的峰相位较下丘脑的延迟。2单色光对松果体生物钟昼夜系统的影响对钟基因昼夜表达的影响:不同单色光照射下,松果体钟基因依然维持明显的昼夜节律性表达,除了红光下cCry2的节律性较白光下明显降低(R2 = 0.243,P = 0.033)。绿光明显增加正调控钟基因的表达量,降低负调控钟基因的表达量;而红光则抑制了正调控钟基因的表达量,增加了负调控钟基因的表达量。与白光相比,绿光下正调控基因的中值和振幅均增加,负调控基因的中值和振幅则减小。在蓝光影响下,正调控钟基因的相位均较白光下发生延迟(0.2 h-0.8 h),负调控钟基因的相位均发生提前(0.1 h-1.5 h)。同时,绿光也明显增加了钟蛋白CLOCK和BMAL1的表达量。对褪黑激素昼夜含量的影响:绿光可以显著提高松果体cAana如基因表达的水平,同时绿光下血浆褪黑激素的含量也高出其他光组1.48%-30.93%(P = 0.000-0.655,ANOVA);而红光下褪黑激素的含量明显较其他光组低了 5.6%-23.4%(P=0.000-0.006,ANOVA)。不同光色下松果体cAanat和血浆褪黑激素均表现出明显的昼夜节律。同时,绿光下松果体cAanat和血浆褪黑激素昼夜振荡的中值和振幅均高于其他光色,而在红光下则均低于其他光组。对松果体视蛋白表达的影响:白光和绿光下,cOpnp的基因表达呈现昼高夜低的表达形式;而蓝光和红光下,cOpnp则呈现出白天低而夜间高的形式。红光下cOpnp的振幅明显下降且昼夜节律性消失(R2= 0.2,P = 0.425)。cOpn4-1基因的表达量呈现白天低夜间高的形式。比起其他光色,绿光下cOpn4-1的昼夜节律性和振幅明显增加。不同单色光下cOpn4-2均呈现白天高夜间低的昼夜节律性表达,其中蓝光下cOpn4-2节律性以及振幅明显降低。在单色光下cCreb的振荡均表现出明显的昼夜节律性。3单色光对下丘脑钟基因昼夜表达的影响不同光色下,下丘脑钟基因均呈现明显的昼夜节律性表达。绿光显著增加了钟基因cClodk、cBmal1以及cCry1的表达量,抑制了cBmal 和cCry2的表达;红光则降低了 cClock、cBmal1以及cCOy1的表达量,却明显增加了 cBBmal2和cCry2的表达量。就节律参数而言,cClock、cBmaal1以及cCry1的中值和振幅在绿光下最大,而cBmal2的中值和振幅在绿光下最小;在红光,cClock和cBmal1的中值和振幅均较白光和绿光小。同时,红光下钟基因峰相位的移动比绿光和蓝光更为明显。对钟蛋白的检测发现,绿光下CLOCK和BMAL1表达的变化与基因水平相类似。同时,钟蛋白CLOCK在下丘脑视交叉上核、前内侧核、室周核、室旁核及正中隆起中均有阳性表达。4松果体褪黑激素介导单色光对下丘脑钟基因表达的影响松果体摘除对血浆褪黑激素昼夜含量的影响:松摘后不同光色下鸡血浆褪黑激素水平与各自假手术组相比均明显下降,但依然保持明显的昼夜节律性;同时绿光下血浆褪黑激素水平仍然高出其他光色 7.0%-55.52%(P= 0.000-0.039,ANOVA)。松果体摘除对下丘脑钟基因表达的影响:随着松摘后褪黑激素水平的下降,在不同光色下下丘钟基因cClock和cPer2的振幅均较各自假手术组明显下降;而cBmal1的中值和振幅较假手术组相似,但红光下和蓝光下的相位分别较各自假手术组延迟2 h和提前2 h。红光下下丘脑cBmal2和cCry2的昼夜表达均失去了节律性。松摘后绿光下cCry1的表达量下降,而红光下表达量升高。松摘后红光下cPer3的表达量依然高于其他光组。同时,下丘脑钟蛋白CLOCK的定位仍然出现在下丘脑视觉视交叉上核、室周核以及室旁核。松果体摘除对下丘脑褪黑激素受体表达的影响:14日龄鸡下丘脑中褪黑激素受体cMel1a、cMel1b和cMel1c均呈现明显的昼夜节律性表达。松摘后三种受体的表达量较对照组和假手术组均分别显著降低 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)。同时,松摘后三种受体的振幅较各自对照组和假手术组明显下降;cMel1b和cMel1c表达的昼夜节律性也随之消失。影响钟基因表达的褪黑激素受体途径:体外添加褪黑激素浓度250 pg/mL时,钟基因cClock的表达量显著高于未添加褪黑激素组45.63%-45.81%(P = 0.003-0.606,ANOVA)。当加入Mella/Mellb非特异性阻断剂Luzindole时,钟基因cClock:的表达量显著下降30.6%-31.4%(P=0.004-0.01,ANOVA);而在仅加入Mellb特异性受体阻断剂4P-PDOT的处理组中,钟基因cClock的表达量比仅添加褪黑激素刺激组的表达量显著低23.6%(P = 0.009,ANOVA);而当仅添加褪黑激素受体Mellc特异性受体阻断剂Prazosin时,钟基因cC/ock的表达量高于其他未加褪黑激素组 44.37%-47.6%(P= 0.002-0.009,ANOVA)。结论:七种核心钟基因在14日龄鸡松果体和下丘脑中的表达均存在明显的昼夜节律性。绿光可以通过影响cOpnp和cOpn4-1来增加松果体正调控钟基因的表达量并降低了负调控钟基因的表达,促进松果体褪黑激素关键合成酶cAanat的表达,从而增加血浆褪黑激素水平。同时,松果体摘除前后下丘脑钟蛋白CLOCK均出现在视觉视交叉上核、室旁核及室周核,单色光可以通过影响松果体褪黑激素的分泌,后者通过其受体Mella和Mellb调节下丘脑钟基因的昼夜节律表达。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S831

【参考文献】