發(fā)布時間：2018-05-24 05:30

  本文選題：單色光 + 脾臟發(fā)育�。� 參考：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：光環(huán)境調(diào)控已成為現(xiàn)代全封閉環(huán)境。下高效家禽養(yǎng)殖的重要手段,直接影響著家禽生產(chǎn)性能和健康。本課題組前期研究發(fā)現(xiàn)在肉雞生長期采用不同單色光照射對肉雞細(xì)胞免疫和體液免疫產(chǎn)生影響,并且與褪黑激素分泌正相關(guān),但其作用機制不清楚。本研究選擇剛出殼的雄性AA肉雞,飼養(yǎng)在不同波長LED光源下(400～700 nm白光、660 nm紅光、560 nm綠光和480 nm藍(lán)光)飼養(yǎng)至42日齡,以褪黑激素為切入點,研究褪黑激素介導(dǎo)單色光影響肉雞脾臟發(fā)育的作用機制。主要研究結(jié)果如下：1、肉雞脾臟發(fā)育、褪黑激素水平及受體表達(dá)的日齡變化7日齡前,肉雞脾臟生長速度大于增重速度,之后則相反。肉雞剛出殼時動脈周圍淋巴鞘(PALS)已出現(xiàn),而直到14日齡脾小結(jié)才出現(xiàn),并隨日齡增長,兩者均快速發(fā)育。采用免疫組織化學(xué)染色對褪黑激素受體(Mel1a, Mel1b和IMel1c)進(jìn)行定位發(fā)現(xiàn)Mel1a分布在紅髓和血管周圍,Mel1b定位在PALS和脾小結(jié),而Mel1c則廣泛定位在紅髓、血管、PALS和脾小結(jié)。褪黑激素受體3亞型(Mel1a、Mel1b和Mel1c)的-nRNA表達(dá)和蛋白呈現(xiàn)年齡依賴性增加。另外,血漿褪黑激素濃度也隨日齡增長而增加,并與脾臟內(nèi)褪黑激素受體(Mel1a, Mel1b和Mel1c)的蛋白變化呈正相關(guān)。2、褪黑激素在單色光影響肉雞脾臟發(fā)育中的作用在14日齡,綠光組肉雞脾臟重量、器官指數(shù)、PALS面積、脾小結(jié)直徑、PCNA陽性細(xì)胞IOD值和T淋巴細(xì)胞增殖活性高于紅光組、白光組和藍(lán)光組(2.71%～30%)(P=0.000～0.989)。同時,綠光較其他光色能顯著提高14日齡肉雞血漿褪黑激素水平(10.93%～19.15%P=0.004～0.029)。松果體摘除使各光色組肉雞血漿褪黑激素含量顯著下降(16.07%～24.11%,P=-0.002～0.011),并伴隨這些脾臟發(fā)育指標(biāo)的不同程度降低(5.84%～30.86%,P=0.000～0.666),且各光色組間差異不顯著(P0.05)。體內(nèi)外研究證實單色綠光通過提高血漿褪黑激素濃度來促進(jìn)肉雞脾臟發(fā)育。3、褪黑激素介導(dǎo)單色綠光促進(jìn)肉雞脾臟T淋巴細(xì)胞增殖的受體途徑14日齡肉雞脾臟內(nèi)褪黑激素受體Mel1b利Mel1c mRNA表達(dá)高于Mel1a。綠光組肉雞脾臟Mel1a和Mel1c mRNA表達(dá)高于白光組(17.86%～40.43%,P=0.004～0.022)和紅光組(22.54%～33.85%,P=0001～0.007);綠光組Mel1bmRNA水平顯著高于白光組、紅光組和藍(lán)組(16.05%～30.56%,P=0.001～0.021)。松果體摘除后,三種褪黑激素受體mRNA水平較對照組相應(yīng)光色顯著下降了42.42%～107.41%(P0.05),且各光色組間差異不顯著(P0.05)。Mel1a, Mellb和Mel1c的蛋白變化與mRNA類似,綠光組Mel1a蛋白顯著高于其他光色組(16.67%～34.62%,P=0.000～0.044)；而綠光組Mel1b和Mel1c蛋白水平高于白光組(19.44%～27.78%,P=0.007～0.009)和¨紅光組(30.3%～39.39%,P=0.000～0.001)。松果體摘除后,三種受體蛋白較對照組相應(yīng)光色下降了37.50%～94.44%(P0.05)。這些結(jié)果顯示單色綠光能較其他光色顯著提高褪黑激素受體的表達(dá)。體外研究結(jié)果顯示,褪黑激素使脾臟T淋巴細(xì)胞增殖活性提高9.7%(P=0.002)。但Mel1b特異性阻斷劑4P-PDOT或Mel1c特異性阻斷劑prazosin卻使褪黑激素誘導(dǎo)的T淋巴細(xì)胞增殖降低了7.60%～11.19%(P=0.000),而Mel1a/Mel1b非特異性阻斷劑luzindol對淋巴細(xì)胞增殖活性無顯著影響(P0.05)。該結(jié)果表明Mel1b和Mel1c可能參與褪黑激素誘導(dǎo)的T淋巴細(xì)胞增殖。4、褪黑激素介導(dǎo)單色綠光促進(jìn)肉雞脾臟T淋巴細(xì)胞增殖的胞內(nèi)信號通路綠光餃紅光利藍(lán)光使14日齡肉雞脾臟ERK1/2活性提高了47.87%～50.17%(P=0.030-0.048)。松果體摘除使綠光組ERK1/2活性較對照組顯著降低40.15%(P=0.012),其他光色組ERK1/2無顯著降低(P=0.178～0.903)。脾臟T淋巴細(xì)胞體外研究結(jié)果顯示,褪黑激素提高了T細(xì)胞增殖活性及胞內(nèi)ERK1/2活性,但MEK-1抑制劑PD98059則能使褪黑激素誘導(dǎo)的T淋巴細(xì)胞增殖活性和胞內(nèi)ERK1/2活性分別下降11.96%(P=0.020)和38.67%(P=0.000)。4P-PDOT和prazosin使褪黑激素誘導(dǎo)的T淋巴細(xì)胞內(nèi)ERK1/2活性降低了38.67%～40%(P=0.000),而luzindole對胞內(nèi)ERK1/2活性無顯著影響。這些結(jié)果表明綠光通過提高褪黑激素水平并由其Mel1b和lMel1c介導(dǎo)激活ERK1/2活性來促進(jìn)肉雞脾臟T淋巴細(xì)胞增殖。另外,AC激動劑forskolin、PKA抑制劑H89、PLC抑制劑或PKC抑制劑使褪黑激素誘導(dǎo)的T淋巴細(xì)胞增殖活性顯著降低17.04%-28.15%(P=0.000-0.001),同時伴隨著胞內(nèi)ERK1-磷酸化水平降低50.55%～70.32%(P=0.000)。這些結(jié)果表明AC/PKA通路和PLC/PKC通路共同激活ERK1/2參與褪黑激素誘導(dǎo)的T淋巴細(xì)胞增殖。5、褪黑激素介導(dǎo)單色光影響肉雞脾臟發(fā)育的非受體途徑綠光組肉雞脾臟SOD、GSH-Px活性和總抗氧化(T-AOC)能力分別比白光組、紅光組和藍(lán)光組高13.35%～22.71%(P=0.000-0.029)、28.92%-35.63%(P=0.000～0.008)和4.25%～6.48%(P=0.001～0.996),相反,綠光組脾臟MDA含量最低,比其他光色低25.28%45.98%(P=0.000)。松果體摘除后,SOD、GSH-Px活性和T-AOC比對照組相應(yīng)光色降低21%～70.01%(P=0.000-0.035),而MDA含量較對照組相應(yīng)光色顯著增加20.47%59.78%(P=0.000～0.002)。這些結(jié)果顯示單色光綠光能顯著促進(jìn)14日齡肉雞脾臟抗氧化酶活性,從而增強肉雞脾臟的抗氧化功能。結(jié)論：在15 lux光照條件下飼養(yǎng)肉雞,單色綠光顯著促進(jìn)脾臟發(fā)育。對其作用機理的研究顯示：在肉雞發(fā)育早期,綠光能顯著提高血漿褪黑激素水平和脾臟褪黑激素受體(Mel1a, Mel1b和Mel1c)表達(dá),褪黑激素或通過Mel1b和Mel1c介導(dǎo)激活A(yù)C/PKA通路和PLC/PKC通路串?dāng)_調(diào)節(jié)ERK1/2活性,或通過增強脾臟抗氧化功能,促進(jìn)脾臟發(fā)育及淋巴細(xì)胞增殖。
[Abstract]:The regulation of light environment has become a modern completely closed environment. The important means of high efficient poultry breeding have a direct impact on the performance and health of poultry. The mechanism was not clear. In this study, the male AA broilers with rigid shell were selected and raised to 42 days of age at different wavelengths of LED (400~700 nm white light, 660 nm red light, 560 nm green light and 480 nm blue light). The mechanism of melatonin mediated the development of the spleen in monochromatic light and shadow chicken was studied with melatonin as the breakthrough point. The main results are as follows: 1 The splenic development of broiler, melatonin level and the day of the expression of the receptor expression were 7 days old. The growth rate of the spleen in broilers was higher than the weight gain speed, then the opposite. The peri arterial lymphatic sheath (PALS) appeared in the chicken just out of the shell, and the splenic nodules only appeared at the age of 14 days, and they both developed rapidly with the age of age. Immunohistochemistry staining was used. The localization of melatonin receptors (Mel1a, Mel1b and IMel1c) found that Mel1a was distributed around the red pulp and blood vessels, Mel1b was located in PALS and splenic nodules, while Mel1c was widely located in the red pulp, blood vessels, PALS and splenic nodules. The -nRNA expression and protein of the melatonin receptor 3 subtype (Mel1a, Mel1b and Mel1c) showed an age dependence. In addition, plasma The concentration of melatonin also increased with the growth of day age, and was positively related to the protein changes of the Mel1a, Mel1b and Mel1c in the spleen. The effect of melatonin on the spleen development of the chicken was 14 days old, the weight of spleen, the number of organs, the area of PALS, the diameter of spleen, the IOD value and T of the PCNA positive cells in the green light group. The lymphocyte proliferation activity was higher than that in red light group, white light group and blue light group (2.71% ~ 30%) (P=0.000 ~ 0.989). At the same time, green light could significantly increase the level of melatonin (10.93% ~ 19.15%P=0.004 ~ 0.029) in plasma of 14 day old broilers. The removal of pineal body melatonin content decreased significantly (16.07% to 24.11%, P=-0). .002 ~ 0.011), with the decrease of the spleen development index (5.84% ~ 30.86%, P=0.000 ~ 0.666), and no significant difference between the light color groups (P0.05). In vitro and in vivo studies have proved that monochromatic green light can promote the development of spleen of broiler by increasing plasma melatonin concentration, and melatonin mediates the T lymphatic lymph of chicken spleen with monochromatic green light. The expression of melatonin receptor Mel1b and Mel1c mRNA in the spleen of 14 day old broilers was higher than that of Mel1a and Mel1c mRNA in chicks of Mel1a. green light group (17.86% ~ 40.43%, P=0.004 to 0.022) and red light group (22.54% to 33.85%, P=0001 to 0.007). The Mel1bmRNA level of the green group was significantly higher than that in the white light group, the red light group and the red light group. Blue Group (16.05% ~ 30.56%, P=0.001 ~ 0.021). After pineal extirpation, three melatonin receptor levels were significantly decreased by 42.42% to 107.41% (P0.05) compared with the control group, and there was no significant difference (P0.05) in each color group (P0.05), and the changes in Mellb and Mel1c were similar to those of mRNA. The Mel1a protein in green group was significantly higher than that of other color groups (16.67). (16.67) The level of Mel1b and Mel1c protein in the green light group was higher than that in the white light group (19.44% ~ 27.78%, P=0.007 ~ 0.009) and red light group (30.3% to 39.39%, P=0.000 to 0.001). After the pineal body extirpation, three receptor proteins decreased by 37.50% to 94.44% (P0.05) compared with the control group. These results showed that the monochromatic green light was better than the others. These results showed that the monochromatic green light was better than the others. In vitro studies showed that melatonin increased the proliferation activity of the spleen T lymphocyte by 9.7% (P=0.002), but the Mel1b specific blocker 4P-PDOT or Mel1c specific blocker prazosin reduced the proliferation of melatonin induced T lymphocytes by 7.60% to 11.19% (P=0.000), and Mel1a/Mel1b The non specific blocker luzindol had no significant effect on the proliferation of lymphocyte (P0.05). The results showed that Mel1b and Mel1c might be involved in the proliferation of T lymphocytes induced by melatonin, and melatonin mediated the intracellular signal of monochromatic green light promoting the proliferation of T lymphocytes in the spleen of broilers, and the red light and blue light of the 14 day old chicken's spleen ER The activity of K1/2 was increased by 47.87% ~ 50.17% (P=0.030-0.048). The extirpation of pineal body made the activity of ERK1/2 in the green light group significantly lower than that of the control group by 40.15% (P=0.012), and the other light color group ERK1/2 had no significant decrease (P=0.178 to 0.903). The results of the splenic T lymphocyte in vitro showed that melatonin increased the proliferation activity and the intracellular ERK1/2 activity of T cells, but MEK-1, but MEK-1, but MEK-1, but MEK-1, but MEK-1 The inhibitor PD98059 could reduce the proliferation activity of T lymphocyte and the intracellular ERK1/2 activity of melatonin by 11.96% (P=0.020) and 38.67% (P=0.000).4P-PDOT and prazosin, which reduced the ERK1/2 activity in the T lymphocytes induced by melatonin by 38.67% to 40% (P=0.000), while luzindole on intracellular ERK1/2 activity had no significant effect. The results show that green light promotes the proliferation of spleen T lymphocytes in chicken by increasing melatonin level and activating ERK1/2 activity mediated by its Mel1b and lMel1c. In addition, AC agonist forskolin, PKA inhibitor H89, PLC inhibitor or PKC inhibitor make the T lymphocyte proliferation activity induced by melatonin significantly lower 17.04%-28.15%. The phosphorylation level of intracellular ERK1- decreased by 50.55% to 70.32% (P=0.000). These results showed that AC/PKA pathway and PLC/PKC pathway co activated ERK1/2 to participate in melatonin induced T lymphocyte proliferation.5. Melatonin mediated the spleen SOD, GSH-Px activity and total resistance of monochromatic light on the spleen development of broilers. T-AOC was 13.35% to 22.71% (P=0.000-0.029), 28.92%-35.63% (P=0.000 ~ 0.008) and 4.25% ~ 6.48% (P=0.001 ~ 0.996) higher than that in white light group and blue light group. On the contrary, the spleen MDA content in the green light group was lowest and 25.28%45.98% (P =0.000) lower than that of other light colors. After the pineal body extirpation, SOD, GSH-Px activity and T-AOC compared with the control group The color decreased by 21% to 70.01% (P=0.000-0.035), while the content of MDA increased significantly by 20.47%59.78% (P=0.000 ~ 0.002) compared with the control group. These results showed that the monochromatic light green light could significantly promote the antioxidant enzyme activity of the spleen of 14 day old broilers, thus enhancing the antioxidant power of the spleen of broilers. Conclusion: raising broilers under the condition of 15 lux light, monochromatic color. Green light significantly promotes spleen development. Research on its mechanism shows that green light can significantly increase the level of melatonin and the expression of melatonin receptor (Mel1a, Mel1b and Mel1c) in the early development of broiler, and melatonin or Mel1b and Mel1c mediated AC/PKA pathway and PLC/PKC pathway crosstalk to regulate ERK1/2 activity, or through Mel1b and PLC/PKC pathway. Enhance the spleen's antioxidant function, promote spleen development and lymphocyte proliferation.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S831
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本文編號：1927857