本文關(guān)鍵詞：SARA狀態(tài)下瘤胃產(chǎn)生的LPS對反芻動物乳腺炎癥反應(yīng)和酪蛋白合成的影響及其調(diào)控　出處：《南京農(nóng)業(yè)大學》2016年博士論文　論文類型：學位論文

  更多相關(guān)文章： SARA LPS 酪蛋白 丁酸鈉 腸系膜灌注 MAC-T

【摘要】：奶業(yè)是我國畜牧業(yè)的重要組成部分,牛奶質(zhì)量安全問題嚴重制約我國奶業(yè)的健康發(fā)展。乳蛋白和乳脂肪是構(gòu)成牛奶營養(yǎng)品質(zhì)的主要物質(zhì)基礎(chǔ),既涉及質(zhì)量安全與消費者的健康,又決定著牛奶的經(jīng)濟價值與核心競爭力。在現(xiàn)代畜牧業(yè)生產(chǎn)中,為了提高奶產(chǎn)量,同時滿足泌乳早期和中期不斷增加的能量需求,奶�；蚰躺窖蛐枰曃垢呔先占Z,這種飼喂方式會引起瘤胃異常發(fā)酵,進而引起亞急性瘤胃酸中毒。瘤胃內(nèi)LPS蓄積并透過胃腸道進入全身循環(huán)系統(tǒng)到達乳腺,到達乳腺的LPS可激活NF- κ B信號通路,抑制mTOR信號通路,導致乳腺炎癥反應(yīng)以及乳蛋白合成功能的抑制。面對我國現(xiàn)有粗飼料品質(zhì)差的現(xiàn)實,如何在目前飼養(yǎng)模式下,既提高動物生產(chǎn)性能,又能維持動物健康,已成為當前高強度飼養(yǎng)中急需解決的問題。丁酸鈉有促進胃腸道組織發(fā)育和增強其屏障功能的作用,能夠抑制LPS介導的NF- κ B信號通路,阻止促炎性細胞因子的釋放,一定程度的治療乳腺的炎癥反應(yīng),恢復(fù)乳腺酪蛋白的合成功能。本試驗還從外源灌注和體外細胞實驗進一步驗證了 LPS對乳腺組織天然免疫反應(yīng)和酪蛋白合成功能的影響。1.消化道產(chǎn)生的LPS對奶牛乳腺免疫功能和乳蛋白合成功能的影響為了滿足泌乳的營養(yǎng)需求,奶牛通常飼喂高精料日糧。但是高精料飼喂會引起亞急性瘤胃酸中毒(SARA),導致乳蛋白下降。本研究旨在探明飼喂高精料日糧的奶牛瘤胃中產(chǎn)生的脂多糖(LPS)對乳腺炎性基因表達和酪蛋白合成的影響。12頭體重為455±28kg的泌乳中期荷斯坦奶牛隨機分為兩組:一組飼喂高精料日糧(含60%的精料和40%粗料,HC); 一組飼喂低精料日糧(含40%的精料和60%粗料,LC),飼喂周期為18周。高精料日糧飼喂后瘤胃PH值小于5.6每天超過3小時,說明高精料日糧成功誘發(fā)了 SARA。我們發(fā)現(xiàn)在高精料日糧飼喂15周以后,乳蛋白顯著下降,體細胞數(shù)顯著升高,且瘤胃液和乳腺動靜脈血漿中的LPS含量顯著升高。乳腺血漿中的促炎性細胞因子IL-1B、IL-6和TNF-α含量顯著升高。通過乳腺組織轉(zhuǎn)錄組芯片技術(shù)的分析,發(fā)現(xiàn)與炎癥相關(guān)的信號通路的表達被激活了,而與酪蛋白合成相關(guān)的信號通路表達受到抑制。同時,高精料組炎癥基因如LBP、TLR4、NF-κB、IL-1B和IL-6等的mRNA表達上調(diào),而αs1酪蛋白和β酪蛋白及其合成相關(guān)基因mTOR、S6K等的表達下調(diào)。本試驗還檢測了相關(guān)信號通路上的關(guān)鍵蛋白表達,發(fā)現(xiàn)其表達與基因的mRNA表達一致,說明了飼喂高精料后,乳腺TLR4介導的炎癥信號通路被激活,酪蛋白合成相關(guān)的mTOR信號通路受到抑制。因此,泌乳奶牛飼喂高精料日糧后,瘤胃產(chǎn)生的LPS增強了乳腺的炎癥基因的表達,抑制了乳蛋白的合成。2. 丁酸鈉對高精料日糧模式下奶山羊乳腺免疫功能和酪蛋白合成功能的影響本試驗采用18頭體重為38.86±2.06 kg泌乳中期的薩能奶山羊,隨機分為三組,每組6只。三組的飼喂方案如下:第一組飼喂低精料日糧,即日糧中含有40%的精料和60%的粗料,精粗比為4:6, LC組;第二組飼喂高精料日糧,精粗比為6:4, HC組;第三組飼喂高精料日糧,精粗比為6:4,并且在日糧中添加丁酸鈉,BHC組。飼喂周期為20周,試驗開始前為所有奶山羊安裝瘤胃瘺管和乳腺動靜脈血管插管,試驗后期采集瘤胃液、奶、乳腺動靜脈血和乳腺組織樣品。高精料日糧引起了瘤胃pH的下降,誘導了 SARA的產(chǎn)生。高精料日糧中添加丁酸鈉后瘤胃pH值有一定回升;HC組外周血中LPS的含量顯著高于LC組和BHC組(p0.01) ; HC組外周血中的促炎性因子IL-1 β、IL-6和TNF-α的含量都顯著高于LC組和BHC組(p0.05 ) ; 丁酸鈉組奶山羊乳腺炎癥基因LBP、TLR4、NF-κB、IL-1B和IL-6等表達相比于高精料組顯著下調(diào)(p0.05),乳蛋白合成相關(guān)基因CSN2、mTOR和S6K等表達顯著上調(diào)(p0.05)。相關(guān)基因的蛋白表達的水平和mRNA水平是一致的,說明丁酸鈉可以抑制LPS介導的TLR4炎癥信號通路,緩解乳蛋白的合成抑制。因此,高精料日糧中添加丁酸鈉可以一定程度上治療高精料日糧誘導的SARA狀態(tài)下的奶山羊乳腺炎癥反應(yīng),恢復(fù)酪蛋白合成功能。3.腸系膜靜脈灌注LPS對奶山羊乳腺天然免疫功能和泌乳功能的影響8只分娩3周以內(nèi)、體重在42 ± 2.8 kg薩能奶山羊,分欄飼喂。試驗中根據(jù)泌奶山羊飼養(yǎng)標準設(shè)計相同的日糧,所有試驗動物均安裝腸系膜血管插管。試驗動物被隨機分為兩組,一組腸系膜灌注LPS作為處理組(Treatment); 一組腸系膜灌注生理鹽水為對照組(Control)。灌注后4h采集血樣,處死后采集乳腺組織樣品。腸系膜灌注LPS的奶山羊血液中的LPS含量遠遠高于灌注生理鹽水的。LPS灌注組的奶山羊乳腺炎癥基因LBP、TLR4、NF-κB、IL-1B和IL-6等表達遠高于對照組,且乳蛋白合成相關(guān)基因CSN2、mTOR,S6K、JAK2和STAT5等表達表達下調(diào)。乳腺中TLR4啟動子區(qū)域染色質(zhì)處于開放狀態(tài),DNA去甲基化,導致TLR4基因表達增強;CSN2啟動子區(qū)域的染色質(zhì)處于壓縮狀態(tài),DNA甲基化率升高,導致CSN2基因表達下調(diào)。本研究表明,腸系膜灌注LPS上調(diào)了乳腺炎性基因的表達,抑制的酪蛋白合成相關(guān)基因的表達,并從一定程度上通過表觀遺傳學機制調(diào)控TLR4和CSN2基因的表達,對乳腺的炎癥反應(yīng)和酪蛋白合成功能造成影響。4. LPS在MAC-T細胞上通過NF- κ B信號通路調(diào)節(jié)炎癥基因的表達本試驗以乳腺上皮細胞傳代細胞系MAC-T細胞為研究對象,分別作5種處理:PBS 組;添加 100ng/mL LPS 組;NF- κ B 過表達組;LPS+BAY11-7085 組和 LPS+MG132組。本試驗發(fā)現(xiàn)LPS濃度在0.1-1000 ng/mL之間時,TLR4表達隨著濃度的升高的增加,LPS濃度大于1000ng/mL后,TLR4表達變化不大。最后我們選擇了 100ng/mL的LPS濃度作為處理濃度。只有當LPS濃度很高時才會導致MAC-T細胞凋亡,因此其凋亡的生物學意義較小。 LPS處理的MAC-T細胞內(nèi)NF-κB表達上調(diào),且促炎性因子的表達也增加了。NF-κB的過表達上調(diào)了細胞的炎癥基因蛋白的表達。NF-κB的兩種抑制劑BAY11-7085和MG132都抑制了 NF- κ B蛋白的表達。BAY11-7085抑制了炎性基因的表達,特別是IL-1B; MG132同樣也抑制了這些基因的表達,尤其是IL-8。因此,我們認為,NF-κB在LPS激活的TLR4炎癥信號通路上具有重要的作用。
[Abstract]:The dairy industry is an important part of animal husbandry in China, the healthy development of milk quality and safety problems seriously restricting China's dairy industry. Milk protein and milk fat is an important material basis for the nutritional quality of milk, which relates to the quality and safety of the health of consumers, but also determines the milk economic value and core competitiveness in modern animal husbandry. In the production, in order to improve milk production, and meet the energy demand of the early and mid lactation dairy cows or goats to fed high concentrate diets, this feeding way will cause abnormal rumen fermentation, thereby causing subacute ruminal acidosis. The rumen LPS accumulation through the gastrointestinal tract into the systemic circulation system to reach the breast. Arrive at the breast LPS can activate NF- B signaling pathway, inhibition of the mTOR signaling pathway, leading to inhibition of inflammation and breast milk protein synthesis function. In the face of China's existing forage The poor quality of the reality, how in the current feeding mode, which can improve the production performance of animal, and to maintain animal health, has become the urgent problems in the breeding of high strength. Sodium butyrate can promote the development and enhance the barrier function of gastrointestinal tissue, NF- can inhibit B signaling pathway mediated by LPS. To prevent the release of pro-inflammatory cytokines, inflammatory reactions in the treatment of breast to a certain extent, the synthesis of functional recovery of casein. This experiment also breast from exogenous perfusion and in vitro experiments to further validate the effect of LPS on anti breast tissue should be natural immunity and casein synthesis function of digestive tract.1. produced LPS of bovine mammary immune function and milk protein synthesis function in order to meet the nutritional needs of lactating cows, usually fed high concentrate diets. But high concentrate feeding can cause subacute ruminal acidosis (SARA), lead Milk protein caused by lipopolysaccharide decline. This study aims at exploring the rumen of dairy cows fed high concentrate diets in (LPS) on the expression of inflammatory genes and casein synthesis.12 head weight was 455 + 28KG in lactating Holstein cows were randomly divided into two groups: one group fed high concentrate diets (containing 60% concentrate and roughage 40%, HC); a group fed low concentrate diets (containing 40% concentrate and 60% coarse material, LC), with a period of 18 weeks. High concentrate diets after rumen pH is less than 5.6 of more than 3 hours a day, the high concentrate diets was successfully induced by SARA. we found that in the high concentrate diet after 15 weeks, milk protein decreased significantly, somatic cell count increased significantly, and LPS content of rumen fluid and breast venous plasma increased significantly. Inflammatory cytokines in plasma of breast IL-1B, IL-6 and TNF- were significantly increased by alpha. Breast tissue transcription group chip technology The analysis showed that the expression of signaling pathways associated with inflammation is activated, and casein synthesis related signaling pathway expression was inhibited. At the same time, the high concentrate group of inflammatory genes such as LBP, TLR4, NF- kappa B, upregulation of IL-1B and IL-6 mRNA, and S1 alpha and beta casein and casein synthesis related gene mTOR, expression of S6K and so on. This experiment also detected key protein related signaling pathway on the expression, we found that the expression and gene expression of mRNA, the fed high concentrate, the inflammatory signaling pathway mediated by activation of breast TLR4, casein synthesis related signaling pathway by mTOR suppression. Therefore, fed high concentrate diets of lactating dairy cows, rumen produced LPS enhanced the expression of inflammatory genes in breast milk protein, inhibit the synthesis of.2. sodium butyrate on high concentrate diets of dairy goat mammary gland model of immune function and casein synthesis 鑳界殑褰卞搷鏈瘯楠岄噰鐢，

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