褪黑激素介导孵化期单色光照射对肉鸡卫星细胞增殖的影响及其信号通路

发布时间：2018-02-28 20:52

  本文关键词： 孵化期 单色光 肉鸡 卫星细胞 褪黑激素 IGF-1　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：本实验室前期研究表明在肉鸡出壳后给予单色光刺激能够影响出壳后肉鸡骨骼肌生长发育。其中单色绿光能够促进肉雏鸡早期(P1-P21日龄)骨骼肌的生长发育,而在发育后期(P21-P49日龄)蓝光的促进效果更为明显。传统胚胎学家认为骨骼肌的发育起于胚胎期,出壳后肌纤维的数目不变。因此我们猜测将光照移至孵化期能否对肉鸡骨骼肌生长发育产生更大的作用?为此,本研究将0胚龄AA肉鸡随机放入红(660nm)、绿(560nm)、蓝(480nm)三种单色光以及复合白光(400～760mn)和黑暗(对照组)条件下孵化,探讨孵化期间不同单色光照射对肉雏鸡骨骼肌发育的影响及作用机制。主要研究结果如下:1.孵化期给予单色光照射对肉雏鸡肌纤维发育与卫星细胞增殖的影响孵化期给予单色绿光照射比其他光组增加肉雏鸡6.7%～8.6%的出壳重,并促进P1、P4、P7和P10日龄肉鸡4.3%～25.2%的体重、9.5%～47.6%胸大肌指数和12.4%～50.0%腓肠肌指数,以及肌纤维面积增加10.1%～103.9%。分离原代卫星细胞试验结果显示,绿光组的卫星细胞相对数量和绝对数量分别高于其他光组6.0%～59.3%和6.6%～77.0%;绿光组胸大肌和腓肠肌中PCNA阳性细胞率分别高于其他光组36.4%～44.5%和35.9%～42.0%。离体MTT方法检测卫星细胞增殖活性,在4个日龄中均显示为绿光组最高,高于其他光组12.5%～61.8%。此外,孵化期间蓝光和白光刺激较黑暗组相比,以上指标均出现不同程度的升高。2.孵化期间单色光影响肉雏鸡卫星细胞增殖分化的分子机制生肌因子:检测5个光组P1、P4、P7和P10日龄肉雏鸡胸大肌和腓肠肌中转录因子--生肌因子转录水平的变化。结果发现孵化期单色绿光照射与其他组相比显著提高骨骼肌中的Pax7(12.36%～45.53%)、Myf5(5.04%～36.99%)、MyoD(6.56%～31.67%)和 Myogenin(11.86%～54.26%)的 mRNA 表达。生长因子:RT-PCR检测P1、P4、P7和P10日龄肉鸡的胸大肌和腓肠肌中正调控生长因子HGF与负调控因子MSTN转录水平,Westenb1ot方法检测c-Met和GDF-8蛋白表达,孵化期单色绿光照射与其他组相比显著提高骨骼肌中的HGF mRNA(16.19%～69.91%)及其受体c-Met的蛋白表达(21.49%～96.43%),延缓MSTN mRNA(11.36%～36.49%)和GDF-8蛋白表达(9.47%～40.88%)。3.IGF-1介导孵化期单色光照射对卫星细胞增殖的作用IGF-1是调控肉鸡骨骼肌卫星细胞增殖的关键生长因子。本研究中,血浆IGF-1水平从P1到P21呈逐渐上升趋势,且绿光组肉雏鸡的血浆IGF-1水平显著高于其他光组7.02%～47.15%,GH作为IGF-1的上游调控激素,同样表现为绿光组肉雏鸡血浆生长激素含量最高,高于其他光组9.39%～59.74%。胸大肌和腓肠肌中IGF-1R的mRNA和蛋白水平变化趋势与血浆IGF-1的一致,均是绿光组最高。Pearson分析显示,血浆IGF-1水平与卫星细胞数(P = 0.001,r=0.989)以及PCNA阳性细胞数(P = 0.001,r = 0.990)显著正相关。体外添加0-200ng/mL浓度IGF-1,引起卫星细胞增殖能力呈剂量依赖性增加,在每剂量组中均表现为绿光的刺激效果最为明显。血浆IGF-1 水平与成肌因子 Pax7(P = 0.004;r = 0.976)、Myf5(P = 0.005;r = 0.973)、MyoD(P = 0.004;r = 0.977)、Myogenin(P = 0.006;r = 0.970)和生长因子 HGFmRNA(P = 0.953;r = 0.012)显著正相关,与MSTNmRNA显著负相关(P = 0.048;r=—0.882),提示孵化期间单色绿光刺激骨骼肌生长过程中,IGF-1通过生肌因子和生长因子调控卫星细胞的增殖。4.IGF-1通过PI3K/Akt通路介导孵化期单色光影响骨骼肌卫星细胞增殖的过程在5个光组中,绿光组肉鸡胸大肌p-Akt表达量最高,p-Akt/t-Akt比值高于其他光组17.10%～60.26%。体外添加100ng/mL外源性IGF-1能够促进卫星细胞增殖和Akt蛋白的活化,添加10μM LY294002(PI3K抑制剂)显著抑制了 IGF-1的促增殖作用。绿光组和黑暗组肉雏鸡胸大肌均注射200μg/200μLLY294002。与对照组(生理盐水)相比,在胸大肌注射LY294002抑制胸大肌中p-Akt表达量(黑暗组和绿光组 p-Akt/t-Akt比值分别下降32.40%和40.31%),降低肉鸡体重(17.39%,黑暗组;28.53%,绿光组)和胸大肌重量(34.54%,黑暗组;39.98%,绿光组)。即孵化期间单色绿光照射能够通过IGF-1胞内信号PI3K/Akt通路调节肉雏鸡骨骼肌卫星细胞的增殖。5.褪黑激素介导孵化期间单色光对肉雏鸡生长发育的影响孵化期单色绿光照射组中P1、P4、P7、P10和P21肉雏鸡血浆褪黑激素水平较其他光组高12.46%～58.61%;单独分析绿光组和黑暗组,发现绿光促进了胚胎后期鸡胚(E15、E18和E21)松果体的发育,提高E18和E21胚龄鸡胚中松果体AANATmRNA表达(12.85%～15.95%)和血浆褪黑激素水平(13.13%～18.20%)。孵化至第八胚龄注射20μg/2μLPACAP6-38(PACAP抑制物)和生理盐水,在黑暗和绿光条件下孵化。结果显示黑暗组和绿光组中P0日龄血浆褪黑激素、GH、IGF-1水平下降,肌纤维面积、大肌纤维比例、骨骼肌PCNA阳性细胞率、卫星细胞数量及增殖活性、Pax7、MRFs(Myf5、MyoD和Myogenin)、HGF和IGF-1R转录水平均出现不同程度的下降,MSTNmRNA上升。与对照组(生理盐水注射组)相比,PACAP6-38注射组绿光组与黑暗组之间的差异消失。体外培养卫星细胞,加入250 pg/mL外源性褪黑激素能显著促进卫星细胞的增殖。这些结果说明褪黑激素在卫星细胞增殖过程中发挥重要作用,并且介导了孵化期单色绿光促进卫星细胞增殖及骨骼肌发育过程。结论:孵化期给予151x单色光刺激,绿光能够显著促进肉雏鸡骨骼肌发育以及卫星细胞增殖能力。孵化期单色绿光照射促进松果体分泌褪黑激素,进而促进生长激素和IGF-1的分泌。IGF-1可以通过与IGF-1R结合激活PI3K/Akt胞内信号通路,促进成肌因子Pax7、Myf5、MyoD、Myogenin和HGF的转录,抑制了负调控生长因子MSTN的转录,促进卫星细胞的增殖,从而促进肌肉发育。
[Abstract]:Our previous studies showed that giving monochromatic stimuli can affect skeletal muscle growth of broiler chickens after hatching development in broilers after hatching. The monochrome green can promote the broiler early (P1-P21 days) the growth and development of skeletal muscle, and in the late development stage (P21-P49 days) Blu ray effect is more obvious. The traditional embryologist think of skeletal muscle development in the embryonic stage, the same number of muscle fibers after hatching. So we will speculate light to the incubation period can on broiler skeletal muscle growth and development have a greater effect? Therefore, this research will be 0 embryo age AA chicken were randomly placed into the red (660nm), green (560nm), blue (480nm) three kinds of monochromatic light and white light composite (400 ~ 760mn) and dark (control group) under the conditions of incubation, Incubation Mechanism during different monochromatic light irradiation effects on broiler skeletal muscle development and function. The main research results are as follows: 1.. The incubation period Give the monochromatic light effect on broiler muscle fiber development and proliferation of satellite cells incubation period given monochrome green radiation increases from 6.7% to 8.6% of the broiler shell weight than other light groups, and promote the P1, P4, P7 and P10 broilers at 4.3% ~ 25.2% weight 9.5% ~ 47.6% pectoralis major index and 12.4% ~ 50% gastrocnemius muscle index and muscle fiber area increased by 10.1% ~ 103.9%. from the primary satellite cell test results show that the relative number of green satellite cells and the absolute number were higher than the other group of 6% light ~ 59.3% and 6.6% ~ 77%; green pectoralis major muscle and gastrocnemius muscle in the ratio of PCNA positive cells were higher than that of the other light group 36.4% ~ 44.5% and 35.9% ~ 42.0%. from the proliferation of satellite cells MTT activity detection method, in 4 days of age were shown as green light was the highest, higher than the other group of 12.5% ~ 61.8%. in addition, the incubation period dark blue and white light stimulation group Compared to the above indexes were increased during the incubation of.2. ring molecular mechanism of broiler monochromatic light satellite cell proliferation and differentiation of myogenic factors in different degree: 5 light detection group P1, P4, changes in the transcription factor myogenic factor transcription level of P7 and P10 day old broiler pectoralis major muscle and gastrocnemius muscle. Found that the incubation period of monochromatic green beam significantly increased in skeletal muscle Pax7 compared with the other groups (12.36% to 45.53%), Myf5 (5.04% ~ 36.99%), MyoD (6.56% ~ 31.67%) and Myogenin (11.86% ~ 54.26%). The expression of mRNA growth factor: RT-PCR P4, P7 and P1 detection, P10 broilers at the chest large muscle and gastrocnemius muscle in the positive regulation of growth factor HGF and negative regulator of the transcription level of MSTN, c-Met and Westenb1ot was used to detect the expression of GDF-8 protein, the incubation period of monochromatic green beam significantly increased in skeletal muscle of HGF mRNA compared with the other groups (16.19% ~ 69.91%) and its receptor c-Met The expression of (21.49% ~ 96.43%), mRNA (11.36% ~ 36.49% MSTN delay) and GDF-8 protein expression (9.47% ~ 40.88%).3.IGF-1 mediated IGF-1 incubation period of monochromatic light irradiation on the proliferation of satellite cells is the key to the proliferation of chicken skeletal muscle satellite cells growth factor. In this study, the plasma level of IGF-1 from P1 to P21 showed a gradual upward trend, the levels of plasma IGF-1 and green broiler was significantly higher than that of other light group 7.02% ~ 47.15%, GH as the IGF-1 upstream regulatory hormones, the same performance as green broiler plasma growth hormone content is the highest, higher than the other group of 9.39% ~ IGF-1R 59.74%. light consistent pectoralis major muscle and gastrocnemius muscle changes in mRNA and the protein level trend and plasma IGF-1, are green was the highest.Pearson analysis showed that the plasma IGF-1 level and the number of satellite cells (P = 0.001, r=0.989) and the number of PCNA positive cells (P = 0.001, r = 0.990) significantly Positive correlation. 0-200ng/mL was added to IGF-1 in vitro, satellite cell proliferation caused a dose-dependent increase in each dose group showed green stimulus effect is most obvious. Plasma levels of IGF-1 and myogenic factor Pax7 (P = 0.004; r = 0.976), Myf5 (P = 0.005; r = 0.973) MyoD, (P = 0.004; r = 0.977), Myogenin (P = 0.006; r = 0.970) and growth factor HGFmRNA (P = 0.953; r = 0.012) had significantly positive correlation, significant negative correlation with MSTNmRNA (P = 0.048; r=, 0.882), suggesting that stimulation of skeletal muscle during incubation monochrome green growth during the process of IGF-1 by myogenic factor and growth factor regulating the proliferation of satellite cells.4.IGF-1 through PI3K/Akt signaling pathway mediated by the incubation period of monochromatic light affects the proliferation of skeletal muscle satellite cells in 5 light group, green group broiler pectoralis major muscle expression of p-Akt was highest, p-Akt/t-Akt ratio was higher than that of the other light group from 17.10% to 60.26%. Adding 100ng/mL exogenous IGF-1 can promote the proliferation of satellite cells and activation of Akt protein, adding 10 M LY294002 (PI3K inhibitor) significantly inhibited the proliferation of IGF-1. Green and dark meat chicken pectoralis major muscle group were injected with 200 g/200 LLY294002. and control group (normal saline) compared to inhibition of expression p-Akt pectoralis major muscle in pectoralis major muscle injection (LY294002 group and p-Akt/t-Akt group of dark green ratio were decreased by 32.40% and 40.31%), reduce the broiler body weight (17.39%, 28.53%, dark group; green group) and the pectoralis major muscle weight (34.54%, 39.98%, dark group; green group). The incubation period can be monochrome green illumination regulation of chicken skeletal muscle satellite cells by IGF-1 intracellular signaling PI3K/Akt pathway in the proliferation of.5. mediated by melatonin during the incubation period of monochromatic light effect on the growth of broiler hatching monochrome green irradiated groups P1, P4, P7, P10 and P21 broilers Chicken plasma melatonin levels than the other group of 12.46% to 58.61% high light and dark green; analysis group group alone, found that green promoted the late embryonic chick embryo (E15, E18 and E21) of the pineal gland development, enhance the expression of E18 and E21 embryonic chick pineal body AANATmRNA (12.85% ~ 15.95%) and plasma melatonin (13.13% ~ 18.20% to eighth). The hatching embryo age injection of 20 mu g/2 mu LPACAP6-38 (PACAP inhibitor) and physiological saline, incubation in the dark and green conditions. The results showed that the dark group and P0 group in the age of green GH, plasma melatonin, IGF-1 level decreased, the area of muscle fiber, muscle fiber ratio, skeletal muscle PCNA positive cells rate, number and proliferation of satellite cells, Pax7, MRFs (Myf5, MyoD and Myogenin), HGF and IGF-1R transcription levels were decreased, different levels of MSTNmRNA increased. And the control group (saline injection group) compared to PACAP6-38 injection group, green group The difference between the group and the darkness disappear. Satellite cells were cultured in vitro, adding 250 pg/mL of exogenous melatonin can significantly promote the proliferation of satellite cells. These results suggest that melatonin plays an important role in the process of proliferation of satellite cells, and mediates the incubation period of monochrome green promote the proliferation of satellite cells and skeletal muscle development process. Conclusion: the incubation period give 151x monochromatic light stimulation, green light could significantly promote the growth of skeletal muscle in broiler chickens and satellite cell proliferation. The incubation period of monochrome green irradiation for the pineal melatonin secretion, and promote growth hormone secretion of.IGF-1 and IGF-1 can be combined with IGF-1R to activate PI3K/Akt intracellular signaling pathways, promote myogenic factors Pax7, Myf5, MyoD. The transcription of Myogenin and HGF, inhibit the negative regulation of transcription factor MSTN promotes the growth and proliferation of satellite cells, thereby promoting muscle growth.

【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
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本文编号：1548912