CD36調控奶山羊乳腺上皮細胞免疫反應的機理研究
發(fā)布時間：2018-08-05 19:15
【摘要】：CD36作為長鏈脂肪酸受體,不但能促進長鏈不飽和脂肪酸的吸收、轉運,而且與其它病原菌模式受體(如TLR4)結合參與到外源或者內源致病性物質的識別、清除并激活下游相關免疫信號通路,但CD36在乳腺中的免疫效能未見報道。本研究旨在對大腸桿菌(E.coli)引起的乳房炎中CD36發(fā)揮的作用及其與TLR4的關系對下游信號通路的影響,并進行長鏈不飽和脂肪酸(如亞麻酸)在LPS誘導的炎癥過程中發(fā)揮的作用研究。本研究克隆了山羊CD36和TLR4序列,通過超表達或干擾CD36以及雙分子熒光互補實驗(BiFC)和免疫沉淀技術(IP)來探討CD36與TLR4在E.coli及LPS中的相互關系以及對下游信號通路的影響。并通過構建CD36脂肪酸結合區(qū)域缺失突變體來研究長鏈不飽和脂肪酸脂肪酸能否通過CD36來發(fā)揮抑制炎癥的功能。本論文獲得以下主要研究結果:1.CD36和TLR4在E.coli引起西農薩能羊乳房炎中發(fā)揮的作用通過組織切片觀測到乳腺腺泡在感染E.coli后的變化,以及炎性細胞的浸潤。首次發(fā)現,與對照組相比在E.coli引起的奶山羊乳房炎中CD36與TLR4的mRNA表達水平顯著升高(p0.01),以及TLR4下游信號通路相關基因的mRNA(如MyD88)和蛋白水平(TRAF6,NF-kB p65,c-JNK,p38-MAPK)也均顯著升高。E.coli引起的奶山羊乳房炎激活CD36和TLR4以及下游信號通路的表達。2.西農薩能羊CD36和TLR4基因的cDNA克隆及相關載體構建成功克隆了西農薩能奶山羊CD36和TLR4基因,并構建了CD36基因的腺病毒超表達載體,將包裝成功的腺病毒Ad-CD36感染乳腺上皮細胞24h后,與空白對照組和感染空病毒組相比CD36的mRNA和蛋白水平有顯著的增加(p0.001)且空白對照組與空病毒組CD36無明顯變化(p0.05)。成功構建了CD36和TLR4的雙分子熒光互補載體pBiFC-VN155-CD36和pBiFC-VC155-TLR4;同時構建了CD36和TLR4的超表達載體pef-NEO-Flag-CD36和pef-NEO-Myc-TLR4。這兩類載體的構建為下一步CD36與TLR4相互作用研究提供了實驗材料。3.CD36參與LPS刺激奶山羊乳腺細胞引起的炎癥反應及下游信號通路的激活通過不同濃度LPS處理乳腺上皮細胞來尋找既能誘導炎癥反應又不引起細胞凋亡或壞死的LPS濃度,用來建立LPS刺激奶山羊乳腺上皮細胞模型。并在該模型基礎上超表達或者干擾CD36來探討CD36對下游信號通路及細胞因子的影響。結果表明在LPS刺激奶山羊乳腺上皮細胞模型中,超表達或者干擾CD36后能激活TLR4/MyD88依賴的信號通路,同時激活下游轉錄因子NF-kB和AP-1的活性,其中AP-1活性的激活是通過c-JNK信號通路而不是p38-MAPK信號通路。并且下游炎性細胞因子除TGF-b外,IL-1β、IL-6、IL-8、TNF-α都會受到CD36超表達或者干擾的影響在LPS處理的細胞中。4.在乳腺上皮細胞中CD36與TLR4相互作用介導E.coli的內化通過BiFC技術初步驗證了在奶山羊乳腺上皮細胞中用E.coli刺激后,CD36與TLR4發(fā)生相互作用,隨后又通過免疫沉淀技術(IP)來再次驗證了E.coli刺激乳腺上皮細胞后CD36與TLR4之間的相互作用。并且通過抗生素保護實驗證明了在奶山羊乳腺上皮細胞中CD36介導E.coli的吞噬作用。5.多不飽和脂肪酸亞麻酸通過CD36抑制LPS誘導的炎癥反應成功構建CD36127-279的過表達載體(Ad-CD36127-279和pef-NEO-Flag-CD36127-279)。發(fā)現了γ-亞麻酸(GLA)而不是亞油酸(LA)可以通過CD36來調節(jié)NF-kB的活性。當干擾CD36或者缺失CD36脂肪酸結合區(qū)域時,GLA在抑制LPS誘導的炎癥中功能將會削弱,包括影響NF-kB的活性級下游炎性因子表達。綜上所述,本研究初步闡明了CD36參與E.coli引起的奶山羊乳房炎引起的炎癥反應,能夠激活下游相關信號通路,在識別、內化E.coli的過程中發(fā)現了CD36與TLR4相互作用。此外,長鏈不飽和脂肪酸是通過CD36來發(fā)揮抑制炎癥的功能。本實驗為奶山羊乳腺免疫功能的調控機理研究提供了理論和實驗依據。
[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.
【學位授予單位】：西北農林科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S827
，
本文編號：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.
【學位授予單位】：西北農林科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S827
，
本文編號：2166734
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