褪黑激素介导单色光影响鸡脾脏T淋巴细胞增殖的作用机制

发布时间：2018-05-24 05:30

  本文选题：单色光 + 脾脏发育　； 参考：《中国农业大学》2016年博士论文

【摘要】：光环境调控已成为现代全封闭环境。下高效家禽养殖的重要手段,直接影响着家禽生产性能和健康。本课题组前期研究发现在肉鸡生长期采用不同单色光照射对肉鸡细胞免疫和体液免疫产生影响,并且与褪黑激素分泌正相关,但其作用机制不清楚。本研究选择刚出壳的雄性AA肉鸡,饲养在不同波长LED光源下(400～700 nm白光、660 nm红光、560 nm绿光和480 nm蓝光)饲养至42日龄,以褪黑激素为切入点,研究褪黑激素介导单色光影响肉鸡脾脏发育的作用机制。主要研究结果如下：1、肉鸡脾脏发育、褪黑激素水平及受体表达的日龄变化7日龄前,肉鸡脾脏生长速度大于增重速度,之后则相反。肉鸡刚出壳时动脉周围淋巴鞘(PALS)已出现,而直到14日龄脾小结才出现,并随日龄增长,两者均快速发育。采用免疫组织化学染色对褪黑激素受体(Mel1a, Mel1b和IMel1c)进行定位发现Mel1a分布在红髓和血管周围,Mel1b定位在PALS和脾小结,而Mel1c则广泛定位在红髓、血管、PALS和脾小结。褪黑激素受体3亚型(Mel1a、Mel1b和Mel1c)的-nRNA表达和蛋白呈现年龄依赖性增加。另外,血浆褪黑激素浓度也随日龄增长而增加,并与脾脏内褪黑激素受体(Mel1a, Mel1b和Mel1c)的蛋白变化呈正相关。2、褪黑激素在单色光影响肉鸡脾脏发育中的作用在14日龄,绿光组肉鸡脾脏重量、器官指数、PALS面积、脾小结直径、PCNA阳性细胞IOD值和T淋巴细胞增殖活性高于红光组、白光组和蓝光组(2.71%～30%)(P=0.000～0.989)。同时,绿光较其他光色能显著提高14日龄肉鸡血浆褪黑激素水平(10.93%～19.15%P=0.004～0.029)。松果体摘除使各光色组肉鸡血浆褪黑激素含量显著下降(16.07%～24.11%,P=-0.002～0.011),并伴随这些脾脏发育指标的不同程度降低(5.84%～30.86%,P=0.000～0.666),且各光色组间差异不显著(P0.05)。体内外研究证实单色绿光通过提高血浆褪黑激素浓度来促进肉鸡脾脏发育。3、褪黑激素介导单色绿光促进肉鸡脾脏T淋巴细胞增殖的受体途径14日龄肉鸡脾脏内褪黑激素受体Mel1b利Mel1c mRNA表达高于Mel1a。绿光组肉鸡脾脏Mel1a和Mel1c mRNA表达高于白光组(17.86%～40.43%,P=0.004～0.022)和红光组(22.54%～33.85%,P=0001～0.007);绿光组Mel1bmRNA水平显著高于白光组、红光组和蓝组(16.05%～30.56%,P=0.001～0.021)。松果体摘除后,三种褪黑激素受体mRNA水平较对照组相应光色显著下降了42.42%～107.41%(P0.05),且各光色组间差异不显著(P0.05)。Mel1a, Mellb和Mel1c的蛋白变化与mRNA类似,绿光组Mel1a蛋白显著高于其他光色组(16.67%～34.62%,P=0.000～0.044)；而绿光组Mel1b和Mel1c蛋白水平高于白光组(19.44%～27.78%,P=0.007～0.009)和¨红光组(30.3%～39.39%,P=0.000～0.001)。松果体摘除后,三种受体蛋白较对照组相应光色下降了37.50%～94.44%(P0.05)。这些结果显示单色绿光能较其他光色显著提高褪黑激素受体的表达。体外研究结果显示,褪黑激素使脾脏T淋巴细胞增殖活性提高9.7%(P=0.002)。但Mel1b特异性阻断剂4P-PDOT或Mel1c特异性阻断剂prazosin却使褪黑激素诱导的T淋巴细胞增殖降低了7.60%～11.19%(P=0.000),而Mel1a/Mel1b非特异性阻断剂luzindol对淋巴细胞增殖活性无显著影响(P0.05)。该结果表明Mel1b和Mel1c可能参与褪黑激素诱导的T淋巴细胞增殖。4、褪黑激素介导单色绿光促进肉鸡脾脏T淋巴细胞增殖的胞内信号通路绿光饺红光利蓝光使14日龄肉鸡脾脏ERK1/2活性提高了47.87%～50.17%(P=0.030-0.048)。松果体摘除使绿光组ERK1/2活性较对照组显著降低40.15%(P=0.012),其他光色组ERK1/2无显著降低(P=0.178～0.903)。脾脏T淋巴细胞体外研究结果显示,褪黑激素提高了T细胞增殖活性及胞内ERK1/2活性,但MEK-1抑制剂PD98059则能使褪黑激素诱导的T淋巴细胞增殖活性和胞内ERK1/2活性分别下降11.96%(P=0.020)和38.67%(P=0.000)。4P-PDOT和prazosin使褪黑激素诱导的T淋巴细胞内ERK1/2活性降低了38.67%～40%(P=0.000),而luzindole对胞内ERK1/2活性无显著影响。这些结果表明绿光通过提高褪黑激素水平并由其Mel1b和lMel1c介导激活ERK1/2活性来促进肉鸡脾脏T淋巴细胞增殖。另外,AC激动剂forskolin、PKA抑制剂H89、PLC抑制剂或PKC抑制剂使褪黑激素诱导的T淋巴细胞增殖活性显著降低17.04%-28.15%(P=0.000-0.001),同时伴随着胞内ERK1-磷酸化水平降低50.55%～70.32%(P=0.000)。这些结果表明AC/PKA通路和PLC/PKC通路共同激活ERK1/2参与褪黑激素诱导的T淋巴细胞增殖。5、褪黑激素介导单色光影响肉鸡脾脏发育的非受体途径绿光组肉鸡脾脏SOD、GSH-Px活性和总抗氧化(T-AOC)能力分别比白光组、红光组和蓝光组高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,绿光组脾脏MDA含量最低,比其他光色低25.28%45.98%(P=0.000)。松果体摘除后,SOD、GSH-Px活性和T-AOC比对照组相应光色降低21%～70.01%(P=0.000-0.035),而MDA含量较对照组相应光色显著增加20.47%59.78%(P=0.000～0.002)。这些结果显示单色光绿光能显著促进14日龄肉鸡脾脏抗氧化酶活性,从而增强肉鸡脾脏的抗氧化功能。结论：在15 lux光照条件下饲养肉鸡,单色绿光显著促进脾脏发育。对其作用机理的研究显示：在肉鸡发育早期,绿光能显著提高血浆褪黑激素水平和脾脏褪黑激素受体(Mel1a, Mel1b和Mel1c)表达,褪黑激素或通过Mel1b和Mel1c介导激活AC/PKA通路和PLC/PKC通路串扰调节ERK1/2活性,或通过增强脾脏抗氧化功能,促进脾脏发育及淋巴细胞增殖。
[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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S831
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