【作者】 张敏；

【导师】 张淑君；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2018， 硕士

【摘要】 世界性多发性的奶牛乳房炎给奶牛健康、奶业经济带来极大的损害,随着人们对乳制品需求量及品质的不断提高,亟待建立防治乳房炎的有效技术。近年来随着分子生物学技术的发展,分子辅助选择已成为遗传改良与育种的重要手段,其重要基础和前提是掌握有效调控奶牛乳房炎的重要基因或重要分子标记。现有研究利用GWAS分析筛选出一个与奶牛乳房炎显著相关的SNP位点,在该位点周围1Mb范围内存在多个脂肪酸结合蛋白FABP(fatty acid binding protein)家族基因。FABP3(fatty acid binding protein-3)脂肪酸结合蛋白3是细胞内重要的脂肪酸转运蛋白,广泛存在于乳腺、心肌等组织中。FABP3通过介导脂肪酸转运参与脂质代谢、能量代谢、免疫调节等各生命活动。因此,本研究以FABP3基因为研究对象,通过构建脂多糖(LPS)诱导奶牛乳腺上皮细胞炎症模型,探讨FABP3对LPS诱导的奶牛乳腺上皮细胞炎症反应的调控及其机制。得到主要结果如下:(1)奶牛白细胞(PBL)和乳腺上皮细胞(bMECs)中FABPs表达水平:在奶牛白细胞(PBL)中,FABP3和FABP5高效表达,FABP4、FABP8、FABP9表达量较低;在奶牛乳腺上皮细胞(bMECs)中,FABP3和FABP4高效表达,FABP5,FABP8和FABP9表达量较低;(2)构建LPS诱导奶牛乳腺上皮细胞(bMECs)炎症模型:确认0.5ug/ml和24h为LPS刺激奶牛乳腺上皮细胞的最佳浓度和时间,且与对照组相比,细胞凋亡水平无明显变化,细胞活性未受影响。即成功构建了LPS刺激乳腺上皮细胞的炎症模型;(3)LPS刺激对奶牛白细胞(PBL)和乳腺上皮细胞(bMECs)中FABP3表达的影响:LPS(0.1ug/ml)诱导奶牛白细胞炎症反应,与对照组相比,3h、9h、12h FABP3的表达水平极显著上升(P<0.01),其中12h表达水平最高;LPS(0.5ug/ml)诱导的奶牛乳腺上皮细胞炎症反应中,与对照组相比,12h、24h FABP3表达极显著上升(P<0.01),24h的表达水平最高;(4)FABP3在LPS诱导的奶牛乳腺上皮细胞(bMECs)炎症中的作用:LPS诱导的奶牛乳腺上皮细胞炎症反应中,siRNA干扰FABP3表达显著抑制炎症因子IL-6和IL-8的表达(P<0.001);通过构建FABP3超表达载体,过表达FABP3极显著促进IL-6和IL-8的表达(P<0.001);同时实验干扰FABP3后,极显著上调过氧化物酶体增殖物激活受体(Peroxisome Proliferator Activated Receptors)PPARα蛋白表达(P<0.001),极显著抑制LPS诱导的NF-κB-p65磷酸化(P<0.001)从而抑制炎症信号通路的激活;(5)不同脂肪酸对FABP3表达及LPS诱导的炎症反应的作用:棕榈酸(饱和脂肪酸)能极显著的促进FABP3的表达(P<0.001),且能极显著促进LPS诱导的炎症因子IL-6和IL-8的表达(P<0.01);而硬脂酸(饱和脂肪酸)对FABP3和炎症因子的表达无显著影响;油酸(不饱和脂肪酸)能显著的抑制FABP3的表达,且能极显著的抑制炎症因子IL-6和IL-8的表达(P<0.01)。综上所述,FABP3在奶牛乳腺上皮细胞(bMECs)和外周血白细胞(PBL)中均高效表达,且LPS有效地诱导细胞中FABP3的表达。干扰FABP3表达可通过上调信号通路中关键基因PPARα表达,抑制炎症信号通路NF-κB激活,从而抑制炎症因子IL-6、IL-8的表达,减轻乳腺上皮细胞炎症反应。不同脂肪酸对FABP3表达与LPS诱导的炎症反应有显著影响,其中油酸(不饱和脂肪酸)能显著的抑制FABP3的表达及细胞炎症反应。本实验结果为研究奶牛乳房炎相关免疫机理、防治奶牛乳房炎提供了新的实验依据和理论参考。更多还原

【Abstract】 The mastitis has made terrible damages for dairy health and industry around the world.As the increasing demonds of quantity and quality,it is crucial to establish effective technology of to prevent and treat mastitis.With the development of molecular biology techniques,marker-assisted selection(MSA)recently has become an important approach for genetic improvement and breeding in cow industry.While MAS depended on the most critical basis that mastitis related genes or molecular markers were known.Genome-wide association study(GWAS)GWAS was an efficient approach to find the significant SNPs associated with dairy cow mastitis.Fatty acid binding protein(FABP)family genes located with 1Mb around the significant SNPs were suggested as the candidate genes.this SNP within 1MB scope.FABP3 is a important fatty acid transporters in cells,and active in multiple tissues such as breast and myocardial.FABP3 can involve in various life activities by mediatng fatty acid transporting,such as lipid metabolism,energy metabolism,immune regulation and so on.In this study,we built LPS-induced cellular inflammation model to research function and mechanism of FABP3 gene on LPS-induced inflammation response in bovine mammary epithelial cells.Experiment results are as follows:(1)The FABPs gene expression level in bovin peripheral blood leucocyte(PBL)and mammary epithelial cells(bMECs):In bovin peripheral blood leucocyte,FABP3 and FABP5 expressed efficiently,but not FABP4,FABP8 or FABP9;in bovine mammary epithelial cells,the expression level of FABP4 and FABP5 is high,but FABP3、FABP8 and FABP9 is low;(2)Built LPS-induced mammary epithelial cells(bMECs)inflammation model:we treated cells with LPS concentration of 0.5ug/ml for 24 h to induce inflammation response.Then the cell viability was measured by flow cytemetry.The result demonstrated LPS failed to influence cell viability,which suggested the model was built successfully;(3)The influence on FABP3 mRNA expression of LPS stimulating in PBL and bMECs: Compared with the control group,LPS increased significantly the mRNA expression of FABP3 at 3-12h(P<0.01),with maximal level at 24 h in PBL.InbMECs,FABP3 mRNA expression with highest level at 24 h was also increased significantly(P<0.01),compared with the control group at 12-24h;(4)The function of FABP3 gene on LPS-induced inflammation response in bMECs: FABP3 gene silencing by siRNA inhibited LPS-induced inflammation factor(IL-6 and IL-8)mRNA expression(P<0.001),and FABP3 overexpression using the FABP3 overexpression plasmid increased IL-6,IL-8 mRNA expression(P<0.001).Therefore we found FABP3 regulated LPS-stimulated crucial inflammation NF-κB passway activation by mediating PPARα(peroxisome proliferator activated receptors)expression;(5)The function of long-chain fatty acid on FABP3 mRNA expression and inflammation response LPS-induced:palmitic acid(saturated fatty acid)can significantly promote the expression of FABP3(P<0.001),and increased the mRNA expression of IL-6 and IL-8 induced by LPS in bMECs(P<0.01);but stearic acid(saturated fatty acid)failed to influence.It is interesting that Oleic acid(unsaturated fatty acid)significantly inhibited FABP3 expression,and expression of the inflammation factor(IL-6and IL-8)(P<0.01).In summary,FABP3 gene significantly expressed in PBL and bMECs,and LPS significantly induced FABP3 mRNA expression.FABP3 gene silencing efficiently inhibited activation of NF-κB passway by increasing PPARα expression,which regulated pro-inflammation factor IL-6 and IL-8 expression to prevent inflammation response.As FABP3 transporting long-chain fatty acid,different long-chain fatty acid influenced the FABP3 mRNA expression and inflammation response in LPS-induced bMECs.The results of our research provided new experimental basis and theory reference for researching on immune mechanism related to mastitis and controling dairy cow mastitis efficiently.更多还原