本文選題：慢性不可預知溫和應激 + 肉雞　； 參考：《中國農業(yè)大學》2017年博士論文

【摘要】：本論文主要研究了慢性不可預知溫和應激對肉雞腸道屏障及免疫功能的影響,并通過小鼠進行進一步驗證;通過日糧添加L-色氨酸研究色氨酸對慢性應激誘導的肉雞腸道屏障及免疫功能的影響。再結合體外試驗,進一步確定L-色氨酸調節(jié)慢性應激誘導的動物腸道屏障結構及免疫功能紊亂的機制。試驗一:為探討色氨酸對慢性不可預知溫和應激(CUMS)肉雞腸道屏障功能的調節(jié)作用,試驗選用320只1日齡愛拔益加AA肉仔雞,隨機分為4個處理,每個處理8個重復,每個重復10只雞。采用玉米-豆粕型基礎日糧,基礎日糧中色氨酸水平為0.21%(0～21日齡)及0.18%(22～42日齡)。然后在基礎日糧中分別添加L-色氨酸,使其分別達到0.42%(0～21日齡)及0.36%(22～42日齡),并分為正常飼養(yǎng)和應激條件下飼養(yǎng),試驗期為6周。應激程序包括:2h黑暗,抓雞,紅或黃旗揮舞101min,陌生人走動101min,驅趕雞群5min,針刺等。結果顯示,CUMS處理顯著降低肉雞生產性能,增加血清皮質酮、β-腎上腺素、去甲腎上腺素水平;日糧添加色氨酸可增加肉雞生產性能,降低應激激素釋放;CUMS處理促進肉雞空腸吲哚胺2,3雙加氧酶(IDO)的基因表達,降低色氨酸羥化酶1(TPH1)的表達,使血清5-羥色胺(5-HT)水平下降;日糧添加色氨酸可明顯提高空腸TPH1的基因表達,抑制IDO基因表達,提高5-HT水平,緩解機體應激反應。另外,日糧添加色氨酸抑制了 CUMS處理誘導的空腸IL-1β、IL-6、TNF-α、IL-8、NF-κB基因表達,降低IL-10表達,提高空腸緊密連接蛋白表達,降低腸道通透性。另,以小鼠為研究對象對上述試驗進一步驗證,試驗結果表明,色氨酸可能是通過增加其代謝酶TPH1的表達,進而提高外周血中5-HT水平,降低應激反應;色氨酸通過抑制腸道促炎癥細胞因子表達,進而提高緊密連接蛋白表達,保障腸道上皮細胞的完整性,維持了正常的腸道屏障功能。試驗二:為探討CUMS處理影響動物腸道屏障功能的機制,揭示L-色氨酸保護腸道屏障免于應激影響的機理。本試驗通過分離培養(yǎng)雞腸道上皮細胞,并利用外源添加地塞米松(DEX)、腎上腺素(AD)、去甲腎上腺素(NE)模擬應激反應,研究上述應激激素對雞腸上皮細胞炎癥相關細胞因子基因表達及緊密連接蛋白的影響。結果發(fā)現(xiàn),應激激素處理不同程度的提高了雞腸道上皮細胞內IL-6、IL-1β、TNF-α等細胞因子的基因表達;且DEX處理顯著降低了雞腸道上皮細胞中緊密連接蛋白ZO-1及Occludin表達;然而,L-色氨酸及應激激素相應受體抑制劑預處理可明顯降低應激激素誘導的細胞因子基因的高表達,提高緊密連接蛋白的表達。另外,本試驗研究了DEX、AD、NE處理對Caco-2細胞跨膜電阻值的影響,結果顯示上述激素處理16、24h后可不同程度的降低Caco-2細胞的跨膜電阻值,而L-色氨酸預處理可緩解應激激素誘導的電阻值下降。上述結果表明,L-色氨酸可能是通過降低應激激素與其相應受體結合,從而削弱激素的效應,降低激素引起的細胞因子的釋放,提高緊密連接蛋白的表達,維持腸道屏障結構的完整性,進而保護腸道屏障功能。試驗三:為探究色氨酸對CUMS處理動物免疫功能的調節(jié)作用,本試驗以肉雞為研究對象并采用小鼠進一步驗證肉雞試驗結果。肉雞試驗設計同試驗一,小鼠試驗設計如下:試驗選用6周齡雄性健康Balb/c小鼠48只,隨機均分為4個處理,每個處理12只。分別是對照組(Ctrl),色'氨酸組(L-Trp),飼喂基礎日糧且應激組(Stress),飼喂高色氨酸日糧且應激組(L-Trp+Stress)。所有小鼠飼養(yǎng)在環(huán)境控制的鼠房內,自由采食和飲水。試驗基礎日糧中色氨酸水平為0.2%。然后在基礎日糧中分別添加L-色氨酸,使其達到0.4%,并分為正常飼養(yǎng)和應激條件下飼養(yǎng),每周記錄小鼠體重,試驗期為8周。小鼠應激程序包括:換籠飼養(yǎng),墊料潮濕,無墊料,冷水浸泡,空水瓶刺激,24h光照/黑暗,黑白顛倒,籠子傾斜45°。結果顯示,CUMS處理降低肉雞新城疫抗體滴度,抑制外周血淋巴細胞的增殖;提高脾臟中應激激素受體的基因表達;日糧添加色氨酸可提高外周血淋巴細胞增殖、NDV抗體滴度,降低脾臟細胞中應激激素受體的基因表達,進而抑制了激素與受體的結合,從而增強機體的免疫機能。以上結果表明,色氨酸可能是通過阻斷應激激素與其受體的結合,從而緩解應激,進而提高機體免疫細胞功能,增強機體免疫機能的。試驗四:為探究CUMS處理影響動物機體免疫機能的機制,揭示L-色氨酸緩解應激調節(jié)免疫機能的機理。本試驗通過小鼠外周血分離獲得T淋巴細胞,并利用外源添加DEX、AD及NE模擬應激反應,研究上述應激激素對T淋巴細胞免疫活性的影響。結果發(fā)現(xiàn),應激激素處理顯著抑制了 T淋巴細胞增殖,而通過對上述應激激素受體特異性抑制則明顯緩解應激激素對T淋巴細胞增殖的影響,表明CUMS處理引起的免疫機能紊亂可能是通過促進了應激激素如皮質酮CORT、NE、AD等激素的釋放及這些應激激素相對應受體在免疫細胞上的表達,進而促進了激素與其受體的結合,增加了激素的敏感性,從而破壞了機體的免疫機能穩(wěn)態(tài)。為進一步揭示L-色氨酸緩解應激反應,調節(jié)機體免疫機能機理。本試驗利用外源添加L-色氨酸及其代謝產物5-HT對T淋巴細胞進行預處理,研究其對應激激素引起的T淋巴細胞活性抑制的影響。結果顯示,L-色氨酸及5-HT預處理均可不同程度的緩解應激激素對T淋巴細胞增殖的影響。L-色氨酸及5-HT可能是通過降低應激激素的釋放及其受體的表達而阻斷二者的結合,從而鈍化應激激素的敏感性,進而保護動物免于應激引起的免疫機能紊亂,而結合體內試驗結果,提示L-色氨酸發(fā)揮作用可能是通過激活了色氨酸代謝途徑中的5-HT代謝途徑,使更多的L-色氨酸分解成為5-HT。綜上所述,L-色氨酸通過降低應激激素的分泌并阻斷應激激素與其受體結合而緩解應激引起的動物腸道屏障結構及免疫紊亂。
[Abstract]:The effect of chronic unpredictable mild stress on intestinal barrier and immune function of broilers was studied in this paper, and the effects of tryptophan on intestinal barrier and immune function induced by chronic stress were studied by adding L- tryptophan in diet. In addition, the L- tryptophan modulation was further determined in vitro. The mechanism of intestinal barrier structure and immune dysfunction induced by chronic stress. Test 1: To investigate the regulatory effect of tryptophan on the intestinal barrier function of chronic unpredictable mild stress (CUMS) broilers, 320 1 day old AA broilers were randomly divided into 4 treatments, each treatment was 8 repetitions, and 10 repeated each. The level of tryptophan in basal diet was 0.21% (0~21 days old) and 0.18% (22~42 days old) with corn soybean meal base diet. Then L- tryptophan was added to basal diet to make it 0.42% (0~21 days old) and 0.36% (22~42 days old) respectively, and were kept under normal feeding and stress conditions for 6 weeks. The stress program was 6 weeks. Including: 2h dark, catch chicken, red or yellow flag waving 101min, strangers walk 101min, drive chicken group 5min, acupuncture and so on. The results showed that CUMS treatment significantly reduced the production performance of broiler, increased serum corticosterone, beta adrenalin, norepinephrine level; dietary tryptophan added to broiler production performance, reduce stress hormone release; CUMS treatment The gene expression of indolamine 2,3 dioxygenase (IDO) was promoted in the jejunum of broiler, and the expression of tryptophan hydroxylase 1 (TPH1) was reduced, and the level of serum 5- hydroxytryptamine (5-HT) decreased. The diet supplemented with tryptophan could obviously improve the gene expression of jejunum TPH1, inhibit the expression of IDO gene, improve the level of 5-HT and alleviate the stress reaction of the body. In addition, the dietary tryptophan added tryptophan to the diet. Inhibiting the expression of IL-1 beta, IL-6, TNF- a, IL-8, NF- kappa B induced by CUMS treatment, reducing the expression of IL-10, improving the expression of the jejunum tight connexin and reducing the permeability of the intestines. 5-HT level in high peripheral blood decreased stress response; tryptophan increased the expression of inflammatory cytokines by inhibiting intestinal proinflammatory cytokines, and then enhanced the expression of tight connexin, guaranteed the integrity of intestinal epithelial cells and maintained normal intestinal barrier function. Test two: To explore the mechanism of CUMS treatment for the intestinal barrier function of the animal and reveal L- tryptophan To protect the intestinal barrier from the effect of stress, this experiment was conducted by separating and cultivating intestinal epithelial cells, and using exogenous dexamethasone (DEX), adrenaline (AD) and norepinephrine (NE) to simulate stress response, and to study the expression of cytokine gene and close connexin in chicken intestinal epithelial cells. The results showed that stress hormone treatment increased the gene expression of IL-6, IL-1 beta, TNF- alpha and other cytokines in the intestinal epithelial cells of chicken, and DEX treatment significantly reduced the expression of closely connexin ZO-1 and Occludin in the intestinal epithelial cells of chicken; however, the pretreatment of L- tryptophan and stress hormone corresponding receptor inhibitors could be obvious. In addition, the effect of DEX, AD and NE on the transmembrane resistance of Caco-2 cells was studied. The results showed that the transmembrane resistance of Caco-2 cells could be reduced to varying degrees after the treatment of 16,24h, and the pretreatment of L- tryptophan could be alleviated. The resistance values induced by stress hormones decrease. The results show that L- tryptophan may reduce the effect of hormone and reduce the release of cytokines caused by hormone, increase the expression of tight connexin, maintain the integrity of the intestinal barrier structure and protect the intestinal barrier function by reducing the hormone effect, reducing the release of cytokines caused by hormone. Test three: in order to explore the effect of tryptophan on the immune function of CUMS in treating animal immune function, this experiment took broiler as the research object and used mice to further verify the results of broiler test. The design of broiler test was the same as the experiment. The experiment was designed as follows: 48 6 weeks male healthy Balb/c mice were selected and divided into 4 treatment randomly, each place was divided into each place. 12 subjects were the control group (Ctrl), the tryptophan acid group (L-Trp), feeding the basal diet and the stress group (Stress), feeding the high tryptophan diet and the stress group (L-Trp+Stress). All the mice were fed in the environment controlled rat room and were free to eat and drink. The level of tryptophan in the basal diet was 0.2%. and then added L- color to the basal diet. Ammonia, which was made up to 0.4%, and kept under normal feeding and stress conditions, recorded the weight of mice weekly for 8 weeks. The stress procedures of mice included cage rearing, wet padding, no cushion, cold water immersion, air water bottle stimulation, 24h light / dark, black and white reverse, and cages tilting 45 degrees. The results showed that CUMS treatment reduced the new antibody antibody to broilers. The titer, inhibiting the proliferation of peripheral blood lymphocyte and improving the gene expression of stress hormone receptor in the spleen, adding tryptophan in the diet can increase the proliferation of peripheral blood lymphocytes, the titer of NDV antibody, reduce the gene expression of stress hormone receptor in the spleen cells, and then inhibit the combination of hormone and receptor, thus enhancing the immune function of the body. The results show that tryptophan may be by blocking the combination of stress hormones and their receptors, thus alleviating stress and improving the immune function of the body and enhancing the immune function of the body. Experiment four: To explore the mechanism of CUMS treatment on the immune function of animal body and reveal the mechanism of L- tryptophan alleviating stress regulating immune function. T lymphocytes were isolated from the peripheral blood of mice, and the effects of stress hormones on the immune activity of T lymphocytes were studied by adding DEX, AD and NE, and the results showed that stress hormone treatment significantly inhibited the proliferation of T lymphocytes, and the stress hormone receptor specific inhibition was significantly relieved by stress. The effect of hormone on the proliferation of T lymphocytes indicates that the immune dysfunction caused by CUMS treatment may be by promoting the release of hormones such as corticosterone, such as corticosterone, CORT, NE, AD, and the expression of these stress hormone relative receptors on the immune cells, thus promoting the combination of hormones with their receptors and increasing the sensitivity of hormones. In order to further reveal the immune function homeostasis of the body, to further reveal the response of L- tryptophan to relieve stress response and regulate the mechanism of immune function, this experiment uses exogenous L- tryptophan and its metabolite 5-HT to pretreat the T lymphocyte, and studies its effect on the inhibition of T lymphatic cell activity induced by stress hormones. The results show that L- tryptophan The effects of acid and 5-HT preconditioning on the proliferation of T lymphocytes can be alleviated to a different degree,.L- tryptophan and 5-HT may be by reducing the release of stress hormones and the expression of their receptors and blocking the combination of the two, thus passivating the sensitivity of stress hormones and thus protecting the animals from immune disorders caused by stress, and the conclusion is that The results of the internal test suggest that L- tryptophan may play a role in activating the 5-HT metabolic pathway in the tryptophan metabolic pathway, making more L- tryptophan decomposed into 5-HT., and L- tryptophan alleviates stress induced intestinal barrier by reducing the secretion of stress hormones and blocking the binding of stress hormones to their receptors. Structural and immune disorders.

【學位授予單位】：中國農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S816

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