CD36调控奶山羊乳腺上皮细胞免疫反应的机理研究
发布时间：2018-08-05 19:15
【摘要】：CD36作为长链脂肪酸受体,不但能促进长链不饱和脂肪酸的吸收、转运,而且与其它病原菌模式受体(如TLR4)结合参与到外源或者内源致病性物质的识别、清除并激活下游相关免疫信号通路,但CD36在乳腺中的免疫效能未见报道。本研究旨在对大肠杆菌(E.coli)引起的乳房炎中CD36发挥的作用及其与TLR4的关系对下游信号通路的影响,并进行长链不饱和脂肪酸(如亚麻酸)在LPS诱导的炎症过程中发挥的作用研究。本研究克隆了山羊CD36和TLR4序列,通过超表达或干扰CD36以及双分子荧光互补实验(BiFC)和免疫沉淀技术(IP)来探讨CD36与TLR4在E.coli及LPS中的相互关系以及对下游信号通路的影响。并通过构建CD36脂肪酸结合区域缺失突变体来研究长链不饱和脂肪酸脂肪酸能否通过CD36来发挥抑制炎症的功能。本论文获得以下主要研究结果:1.CD36和TLR4在E.coli引起西农萨能羊乳房炎中发挥的作用通过组织切片观测到乳腺腺泡在感染E.coli后的变化,以及炎性细胞的浸润。首次发现,与对照组相比在E.coli引起的奶山羊乳房炎中CD36与TLR4的mRNA表达水平显著升高(p0.01),以及TLR4下游信号通路相关基因的mRNA(如MyD88)和蛋白水平(TRAF6,NF-kB p65,c-JNK,p38-MAPK)也均显著升高。E.coli引起的奶山羊乳房炎激活CD36和TLR4以及下游信号通路的表达。2.西农萨能羊CD36和TLR4基因的cDNA克隆及相关载体构建成功克隆了西农萨能奶山羊CD36和TLR4基因,并构建了CD36基因的腺病毒超表达载体,将包装成功的腺病毒Ad-CD36感染乳腺上皮细胞24h后,与空白对照组和感染空病毒组相比CD36的mRNA和蛋白水平有显著的增加(p0.001)且空白对照组与空病毒组CD36无明显变化(p0.05)。成功构建了CD36和TLR4的双分子荧光互补载体pBiFC-VN155-CD36和pBiFC-VC155-TLR4;同时构建了CD36和TLR4的超表达载体pef-NEO-Flag-CD36和pef-NEO-Myc-TLR4。这两类载体的构建为下一步CD36与TLR4相互作用研究提供了实验材料。3.CD36参与LPS刺激奶山羊乳腺细胞引起的炎症反应及下游信号通路的激活通过不同浓度LPS处理乳腺上皮细胞来寻找既能诱导炎症反应又不引起细胞凋亡或坏死的LPS浓度,用来建立LPS刺激奶山羊乳腺上皮细胞模型。并在该模型基础上超表达或者干扰CD36来探讨CD36对下游信号通路及细胞因子的影响。结果表明在LPS刺激奶山羊乳腺上皮细胞模型中,超表达或者干扰CD36后能激活TLR4/MyD88依赖的信号通路,同时激活下游转录因子NF-kB和AP-1的活性,其中AP-1活性的激活是通过c-JNK信号通路而不是p38-MAPK信号通路。并且下游炎性细胞因子除TGF-b外,IL-1β、IL-6、IL-8、TNF-α都会受到CD36超表达或者干扰的影响在LPS处理的细胞中。4.在乳腺上皮细胞中CD36与TLR4相互作用介导E.coli的内化通过BiFC技术初步验证了在奶山羊乳腺上皮细胞中用E.coli刺激后,CD36与TLR4发生相互作用,随后又通过免疫沉淀技术(IP)来再次验证了E.coli刺激乳腺上皮细胞后CD36与TLR4之间的相互作用。并且通过抗生素保护实验证明了在奶山羊乳腺上皮细胞中CD36介导E.coli的吞噬作用。5.多不饱和脂肪酸亚麻酸通过CD36抑制LPS诱导的炎症反应成功构建CD36127-279的过表达载体(Ad-CD36127-279和pef-NEO-Flag-CD36127-279)。发现了γ-亚麻酸(GLA)而不是亚油酸(LA)可以通过CD36来调节NF-kB的活性。当干扰CD36或者缺失CD36脂肪酸结合区域时,GLA在抑制LPS诱导的炎症中功能将会削弱,包括影响NF-kB的活性级下游炎性因子表达。综上所述,本研究初步阐明了CD36参与E.coli引起的奶山羊乳房炎引起的炎症反应,能够激活下游相关信号通路,在识别、内化E.coli的过程中发现了CD36与TLR4相互作用。此外,长链不饱和脂肪酸是通过CD36来发挥抑制炎症的功能。本实验为奶山羊乳腺免疫功能的调控机理研究提供了理论和实验依据。
[Abstract]:CD36, as a long chain fatty acid receptor, not only promotes the absorption and transport of long chain unsaturated fatty acids, but also participates in the identification of exogenous or endogenous pathogenic substances by combining with other pathogenic bacteria model receptors (such as TLR4), scavenging and activating the downstream related immune signaling pathways, but the immune efficacy of CD36 in the breast is not reported. This study is aimed at this study. The role of CD36 in the E.coli induced mastitis and the effect of the relationship with TLR4 on the downstream signal pathway and the role of long chain unsaturated fatty acids such as linolenic acid (linolenic acid) in the process of LPS induced inflammation. This study cloned the CD36 and TLR4 sequences of goats, overexpressed or interfered with CD36 and bipartite. The relationship between CD36 and TLR4 (BiFC) and immunoprecipitation technique (IP) to explore the relationship between CD36 and TLR4 in E.coli and LPS and the effect on the downstream signal pathway. And by constructing the CD36 fatty acid binding regional deletion mutants to study whether the long chain fatty acid fatty acids can inhibit inflammation through CD36. The main results are as follows: the role of 1.CD36 and TLR4 in E.coli induced mastitis in Sinon Sam sheep was observed by tissue section, and the changes in the mammary gland after infection of E.coli and the infiltration of inflammatory cells were observed. For the first time, the level of mRNA expression of CD36 and TLR4 in dairy goat mastitis caused by E.coli was significantly higher than that of the control group. Increase (P0.01), as well as mRNA (such as MyD88) and protein level (TRAF6, NF-kB p65, c-JNK, p38-MAPK) of the downstream signal pathway related genes (TRAF6, NF-kB p65, c-JNK, p38-MAPK) also significantly increased the activation of CD36, TLR4, and downstream signaling pathways in milk goat mastitis. The CD36 and TLR4 genes of Sinong sappan milk goat were augmentation, and the adenovirus overexpression vector of CD36 gene was constructed. After the successful packaging of adenovirus Ad-CD36 infected the breast epithelial cells 24h, the mRNA and protein levels of CD36 in the blank control group and the infected empty virus group were significantly increased (p0.001), and the blank control group and the empty virus group had no CD36. A significant change (P0.05). A successful construction of CD36 and TLR4 bimolecular fluorescent complementary carriers pBiFC-VN155-CD36 and pBiFC-VC155-TLR4, and the construction of the two carriers of CD36 and TLR4 overexpression vectors pef-NEO-Flag-CD36 and pef-NEO-Myc-TLR4. provide experimental materials for the next step of CD36 and TLR4. The inflammatory response and the activation of the downstream signal pathway in the mammary gland cells of the milk goats are treated with different concentrations of LPS in the breast epithelial cells to find the LPS concentration that can induce the inflammatory response and not cause apoptosis or necrosis. It is used to establish the LPS stimulation of the mammary epithelial cell model of milk goats. CD36 was used to investigate the effect of CD36 on the downstream signal pathway and cytokine. The results showed that in the LPS stimulated breast epithelial cell model of milk goats, after the overexpression or interference of CD36, the TLR4/MyD88 dependent signaling pathway was activated and the activity of the downstream transcription factor NF-kB and AP-1 was activated. The activation of AP-1 activity was through the c-JNK signaling pathway. Not p38-MAPK signaling pathway, and downstream inflammatory cytokines except TGF-b, IL-1 beta, IL-6, IL-8, TNF- alpha are all affected by CD36 overexpression or interference in LPS treated cells,.4. in mammary epithelial cells mediated E.coli is mediated by CD36 and TLR4 in breast epithelial cells. The interaction between CD36 and TLR4 was stimulated with E.coli, and then the interaction between CD36 and TLR4 after E.coli stimulation of mammary epithelial cells was subsequently confirmed by immunoprecipitation (IP). And through the antibiotic protection experiment, it was proved that CD36 mediates the phagocytosis of.5. polyunsaturated fat in the mammary epithelial cells of dairy goats. Acid linolenic acid successfully constructed CD36127-279 overexpression vector (Ad-CD36127-279 and pef-NEO-Flag-CD36127-279) by inhibiting the inflammatory response induced by LPS by CD36. It was found that gamma linolenic acid (GLA), not linoleic acid (LA), could regulate the activity of NF-kB through CD36. When the CD36 or the CD36 fatty acid binding area was absent, GLA was inhibited. The function of the inflammation in the guide will be weakened, including the expression of the downstream inflammatory factors affecting the active grade of NF-kB. In summary, this study preliminarily clarified the inflammatory response caused by CD36's involvement in dairy goat mastitis caused by E.coli, and could activate the downstream related signaling pathways. The interaction of CD36 and TLR4 was found in the identification and internalization of E.coli. In addition, long chain unsaturated fatty acids play a role in inhibiting inflammation through CD36. This experiment provides a theoretical and experimental basis for the study of the mechanism of mammary immune function in dairy goats.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S827
，
本文编号：2166734
[Abstract]:CD36, as a long chain fatty acid receptor, not only promotes the absorption and transport of long chain unsaturated fatty acids, but also participates in the identification of exogenous or endogenous pathogenic substances by combining with other pathogenic bacteria model receptors (such as TLR4), scavenging and activating the downstream related immune signaling pathways, but the immune efficacy of CD36 in the breast is not reported. This study is aimed at this study. The role of CD36 in the E.coli induced mastitis and the effect of the relationship with TLR4 on the downstream signal pathway and the role of long chain unsaturated fatty acids such as linolenic acid (linolenic acid) in the process of LPS induced inflammation. This study cloned the CD36 and TLR4 sequences of goats, overexpressed or interfered with CD36 and bipartite. The relationship between CD36 and TLR4 (BiFC) and immunoprecipitation technique (IP) to explore the relationship between CD36 and TLR4 in E.coli and LPS and the effect on the downstream signal pathway. And by constructing the CD36 fatty acid binding regional deletion mutants to study whether the long chain fatty acid fatty acids can inhibit inflammation through CD36. The main results are as follows: the role of 1.CD36 and TLR4 in E.coli induced mastitis in Sinon Sam sheep was observed by tissue section, and the changes in the mammary gland after infection of E.coli and the infiltration of inflammatory cells were observed. For the first time, the level of mRNA expression of CD36 and TLR4 in dairy goat mastitis caused by E.coli was significantly higher than that of the control group. Increase (P0.01), as well as mRNA (such as MyD88) and protein level (TRAF6, NF-kB p65, c-JNK, p38-MAPK) of the downstream signal pathway related genes (TRAF6, NF-kB p65, c-JNK, p38-MAPK) also significantly increased the activation of CD36, TLR4, and downstream signaling pathways in milk goat mastitis. The CD36 and TLR4 genes of Sinong sappan milk goat were augmentation, and the adenovirus overexpression vector of CD36 gene was constructed. After the successful packaging of adenovirus Ad-CD36 infected the breast epithelial cells 24h, the mRNA and protein levels of CD36 in the blank control group and the infected empty virus group were significantly increased (p0.001), and the blank control group and the empty virus group had no CD36. A significant change (P0.05). A successful construction of CD36 and TLR4 bimolecular fluorescent complementary carriers pBiFC-VN155-CD36 and pBiFC-VC155-TLR4, and the construction of the two carriers of CD36 and TLR4 overexpression vectors pef-NEO-Flag-CD36 and pef-NEO-Myc-TLR4. provide experimental materials for the next step of CD36 and TLR4. The inflammatory response and the activation of the downstream signal pathway in the mammary gland cells of the milk goats are treated with different concentrations of LPS in the breast epithelial cells to find the LPS concentration that can induce the inflammatory response and not cause apoptosis or necrosis. It is used to establish the LPS stimulation of the mammary epithelial cell model of milk goats. CD36 was used to investigate the effect of CD36 on the downstream signal pathway and cytokine. The results showed that in the LPS stimulated breast epithelial cell model of milk goats, after the overexpression or interference of CD36, the TLR4/MyD88 dependent signaling pathway was activated and the activity of the downstream transcription factor NF-kB and AP-1 was activated. The activation of AP-1 activity was through the c-JNK signaling pathway. Not p38-MAPK signaling pathway, and downstream inflammatory cytokines except TGF-b, IL-1 beta, IL-6, IL-8, TNF- alpha are all affected by CD36 overexpression or interference in LPS treated cells,.4. in mammary epithelial cells mediated E.coli is mediated by CD36 and TLR4 in breast epithelial cells. The interaction between CD36 and TLR4 was stimulated with E.coli, and then the interaction between CD36 and TLR4 after E.coli stimulation of mammary epithelial cells was subsequently confirmed by immunoprecipitation (IP). And through the antibiotic protection experiment, it was proved that CD36 mediates the phagocytosis of.5. polyunsaturated fat in the mammary epithelial cells of dairy goats. Acid linolenic acid successfully constructed CD36127-279 overexpression vector (Ad-CD36127-279 and pef-NEO-Flag-CD36127-279) by inhibiting the inflammatory response induced by LPS by CD36. It was found that gamma linolenic acid (GLA), not linoleic acid (LA), could regulate the activity of NF-kB through CD36. When the CD36 or the CD36 fatty acid binding area was absent, GLA was inhibited. The function of the inflammation in the guide will be weakened, including the expression of the downstream inflammatory factors affecting the active grade of NF-kB. In summary, this study preliminarily clarified the inflammatory response caused by CD36's involvement in dairy goat mastitis caused by E.coli, and could activate the downstream related signaling pathways. The interaction of CD36 and TLR4 was found in the identification and internalization of E.coli. In addition, long chain unsaturated fatty acids play a role in inhibiting inflammation through CD36. This experiment provides a theoretical and experimental basis for the study of the mechanism of mammary immune function in dairy goats.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S827
，
本文编号：2166734
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