本文關(guān)鍵詞： 孵化期 單色光 肉雞 衛(wèi)星細(xì)胞 褪黑激素 IGF-1　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：本實(shí)驗(yàn)室前期研究表明在肉雞出殼后給予單色光刺激能夠影響出殼后肉雞骨骼肌生長(zhǎng)發(fā)育。其中單色綠光能夠促進(jìn)肉雛雞早期(P1-P21日齡)骨骼肌的生長(zhǎng)發(fā)育,而在發(fā)育后期(P21-P49日齡)藍(lán)光的促進(jìn)效果更為明顯。傳統(tǒng)胚胎學(xué)家認(rèn)為骨骼肌的發(fā)育起于胚胎期,出殼后肌纖維的數(shù)目不變。因此我們猜測(cè)將光照移至孵化期能否對(duì)肉雞骨骼肌生長(zhǎng)發(fā)育產(chǎn)生更大的作用?為此,本研究將0胚齡AA肉雞隨機(jī)放入紅(660nm)、綠(560nm)、藍(lán)(480nm)三種單色光以及復(fù)合白光(400～760mn)和黑暗(對(duì)照組)條件下孵化,探討孵化期間不同單色光照射對(duì)肉雛雞骨骼肌發(fā)育的影響及作用機(jī)制。主要研究結(jié)果如下:1.孵化期給予單色光照射對(duì)肉雛雞肌纖維發(fā)育與衛(wèi)星細(xì)胞增殖的影響孵化期給予單色綠光照射比其他光組增加肉雛雞6.7%～8.6%的出殼重,并促進(jìn)P1、P4、P7和P10日齡肉雞4.3%～25.2%的體重、9.5%～47.6%胸大肌指數(shù)和12.4%～50.0%腓腸肌指數(shù),以及肌纖維面積增加10.1%～103.9%。分離原代衛(wèi)星細(xì)胞試驗(yàn)結(jié)果顯示,綠光組的衛(wèi)星細(xì)胞相對(duì)數(shù)量和絕對(duì)數(shù)量分別高于其他光組6.0%～59.3%和6.6%～77.0%;綠光組胸大肌和腓腸肌中PCNA陽(yáng)性細(xì)胞率分別高于其他光組36.4%～44.5%和35.9%～42.0%。離體MTT方法檢測(cè)衛(wèi)星細(xì)胞增殖活性,在4個(gè)日齡中均顯示為綠光組最高,高于其他光組12.5%～61.8%。此外,孵化期間藍(lán)光和白光刺激較黑暗組相比,以上指標(biāo)均出現(xiàn)不同程度的升高。2.孵化期間單色光影響肉雛雞衛(wèi)星細(xì)胞增殖分化的分子機(jī)制生肌因子:檢測(cè)5個(gè)光組P1、P4、P7和P10日齡肉雛雞胸大肌和腓腸肌中轉(zhuǎn)錄因子--生肌因子轉(zhuǎn)錄水平的變化。結(jié)果發(fā)現(xiàn)孵化期單色綠光照射與其他組相比顯著提高骨骼肌中的Pax7(12.36%～45.53%)、Myf5(5.04%～36.99%)、MyoD(6.56%～31.67%)和 Myogenin(11.86%～54.26%)的 mRNA 表達(dá)。生長(zhǎng)因子:RT-PCR檢測(cè)P1、P4、P7和P10日齡肉雞的胸大肌和腓腸肌中正調(diào)控生長(zhǎng)因子HGF與負(fù)調(diào)控因子MSTN轉(zhuǎn)錄水平,Westenb1ot方法檢測(cè)c-Met和GDF-8蛋白表達(dá),孵化期單色綠光照射與其他組相比顯著提高骨骼肌中的HGF mRNA(16.19%～69.91%)及其受體c-Met的蛋白表達(dá)(21.49%～96.43%),延緩MSTN mRNA(11.36%～36.49%)和GDF-8蛋白表達(dá)(9.47%～40.88%)。3.IGF-1介導(dǎo)孵化期單色光照射對(duì)衛(wèi)星細(xì)胞增殖的作用IGF-1是調(diào)控肉雞骨骼肌衛(wèi)星細(xì)胞增殖的關(guān)鍵生長(zhǎng)因子。本研究中,血漿IGF-1水平從P1到P21呈逐漸上升趨勢(shì),且綠光組肉雛雞的血漿IGF-1水平顯著高于其他光組7.02%～47.15%,GH作為IGF-1的上游調(diào)控激素,同樣表現(xiàn)為綠光組肉雛雞血漿生長(zhǎng)激素含量最高,高于其他光組9.39%～59.74%。胸大肌和腓腸肌中IGF-1R的mRNA和蛋白水平變化趨勢(shì)與血漿IGF-1的一致,均是綠光組最高。Pearson分析顯示,血漿IGF-1水平與衛(wèi)星細(xì)胞數(shù)(P = 0.001,r=0.989)以及PCNA陽(yáng)性細(xì)胞數(shù)(P = 0.001,r = 0.990)顯著正相關(guān)。體外添加0-200ng/mL濃度IGF-1,引起衛(wèi)星細(xì)胞增殖能力呈劑量依賴性增加,在每劑量組中均表現(xiàn)為綠光的刺激效果最為明顯。血漿IGF-1 水平與成肌因子 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)和生長(zhǎng)因子 HGFmRNA(P = 0.953;r = 0.012)顯著正相關(guān),與MSTNmRNA顯著負(fù)相關(guān)(P = 0.048;r=—0.882),提示孵化期間單色綠光刺激骨骼肌生長(zhǎng)過(guò)程中,IGF-1通過(guò)生肌因子和生長(zhǎng)因子調(diào)控衛(wèi)星細(xì)胞的增殖。4.IGF-1通過(guò)PI3K/Akt通路介導(dǎo)孵化期單色光影響骨骼肌衛(wèi)星細(xì)胞增殖的過(guò)程在5個(gè)光組中,綠光組肉雞胸大肌p-Akt表達(dá)量最高,p-Akt/t-Akt比值高于其他光組17.10%～60.26%。體外添加100ng/mL外源性IGF-1能夠促進(jìn)衛(wèi)星細(xì)胞增殖和Akt蛋白的活化,添加10μM LY294002(PI3K抑制劑)顯著抑制了 IGF-1的促增殖作用。綠光組和黑暗組肉雛雞胸大肌均注射200μg/200μLLY294002。與對(duì)照組(生理鹽水)相比,在胸大肌注射LY294002抑制胸大肌中p-Akt表達(dá)量(黑暗組和綠光組 p-Akt/t-Akt比值分別下降32.40%和40.31%),降低肉雞體重(17.39%,黑暗組;28.53%,綠光組)和胸大肌重量(34.54%,黑暗組;39.98%,綠光組)。即孵化期間單色綠光照射能夠通過(guò)IGF-1胞內(nèi)信號(hào)PI3K/Akt通路調(diào)節(jié)肉雛雞骨骼肌衛(wèi)星細(xì)胞的增殖。5.褪黑激素介導(dǎo)孵化期間單色光對(duì)肉雛雞生長(zhǎng)發(fā)育的影響孵化期單色綠光照射組中P1、P4、P7、P10和P21肉雛雞血漿褪黑激素水平較其他光組高12.46%～58.61%;單獨(dú)分析綠光組和黑暗組,發(fā)現(xiàn)綠光促進(jìn)了胚胎后期雞胚(E15、E18和E21)松果體的發(fā)育,提高E18和E21胚齡雞胚中松果體AANATmRNA表達(dá)(12.85%～15.95%)和血漿褪黑激素水平(13.13%～18.20%)。孵化至第八胚齡注射20μg/2μLPACAP6-38(PACAP抑制物)和生理鹽水,在黑暗和綠光條件下孵化。結(jié)果顯示黑暗組和綠光組中P0日齡血漿褪黑激素、GH、IGF-1水平下降,肌纖維面積、大肌纖維比例、骨骼肌PCNA陽(yáng)性細(xì)胞率、衛(wèi)星細(xì)胞數(shù)量及增殖活性、Pax7、MRFs(Myf5、MyoD和Myogenin)、HGF和IGF-1R轉(zhuǎn)錄水平均出現(xiàn)不同程度的下降,MSTNmRNA上升。與對(duì)照組(生理鹽水注射組)相比,PACAP6-38注射組綠光組與黑暗組之間的差異消失。體外培養(yǎng)衛(wèi)星細(xì)胞,加入250 pg/mL外源性褪黑激素能顯著促進(jìn)衛(wèi)星細(xì)胞的增殖。這些結(jié)果說(shuō)明褪黑激素在衛(wèi)星細(xì)胞增殖過(guò)程中發(fā)揮重要作用,并且介導(dǎo)了孵化期單色綠光促進(jìn)衛(wèi)星細(xì)胞增殖及骨骼肌發(fā)育過(guò)程。結(jié)論:孵化期給予151x單色光刺激,綠光能夠顯著促進(jìn)肉雛雞骨骼肌發(fā)育以及衛(wèi)星細(xì)胞增殖能力。孵化期單色綠光照射促進(jìn)松果體分泌褪黑激素,進(jìn)而促進(jìn)生長(zhǎng)激素和IGF-1的分泌。IGF-1可以通過(guò)與IGF-1R結(jié)合激活PI3K/Akt胞內(nèi)信號(hào)通路,促進(jìn)成肌因子Pax7、Myf5、MyoD、Myogenin和HGF的轉(zhuǎn)錄,抑制了負(fù)調(diào)控生長(zhǎng)因子MSTN的轉(zhuǎn)錄,促進(jìn)衛(wèi)星細(xì)胞的增殖,從而促進(jìn)肌肉發(fā)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.

【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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本文編號(hào)：1548912