SIGIRR对IL-1R/TLRs信号通路的负性调控作用研究进展

单免疫球蛋白白介素1受体相关蛋白（SIGIRR）是TIR超家族成员,最新研究表明它可负性调节IL-1R/TLRs受体介导的固有免疫应答,且在感染性疾病、肿瘤及自体免疫病诱导的炎症反应调节上所起的重要作用已经被阐明。作者主要综述了SIGIRR基因和蛋白表达模式在人、小鼠、牛等物种间的差异性,因其结构特异性所具有的对IL-1R/TLRs炎性信号通路的负性调控和具体的调控机理,以及在不同炎性相关疾病的发生和发展中抑制炎性反应的机制,进而为临床的炎性相关疾病治疗提供理论借鉴,同时也拓宽了SIGIRR基因在动物疾病治疗中的应用范围。     

β-羟基丁酸通过激活TLR4/NF-κB通路诱导奶牛乳腺上皮细胞炎性反应

旨在探究Toll样受体4(Toll-like receptor 4,TLR4)/核转录因子kappaB(nuclear factor-kappaB,NF-κB)通路在β-羟基丁酸(β-hydroxybutyrate, BHBA)诱导奶牛乳腺上皮细胞炎性反应的分子作用机制。通过添加不同浓度的BHBA诱导奶牛乳腺上皮细胞MAC-T以模拟奶牛高酮血症中乳腺上皮细胞的炎性反应,并利用RNAi技术将TLR4-siRNA转染细胞42 h后加入3.6 mmol/L BHBA再处理24 h;通过RT-qPCR检测肿瘤坏死因子-α(tumor necrosis factor-alpha, TNF-α)、白介素-1β(interleukin-1beta, IL-1β)和IL-6等炎性因子的表达变化,并采用Western blot法测定TLR4/NF-κB通路关键蛋白的相对表达水平。结果表明,与对照组相比,不同浓度BHBA处理后,乳腺上皮细胞中TNF-α、IL-1β和IL-6 mRNA水平升高,当BHBA浓度为1.2 mmol/L或以上时,p-NF-κB p65/NF-κB p65比值和TRAF6蛋白水平显...     

LPS诱导的奶牛子宫内膜细胞TLR4信号传导通路研究概况

子宫内膜炎是奶牛产后细菌感染子宫而引起的产科疾病,严重影响奶牛业的健康发展。革兰阴性细菌中的大肠埃希菌是引起奶牛子宫内膜炎的主要致病菌,细菌脂多糖(lipopolysaccharide,LPS)是革兰阴性细菌细胞壁的重要组成成分,也是近年研究奶牛子宫内膜炎发病机制的重要诱导物。论文综述了LPS、Toll样受体(toll-like receptors,TLRs)及炎性因子,初步剖析了LPS诱导的炎症反应信号传导通路TLR4信号通路,为奶牛子宫内膜炎的研究提供参考。     

必需氨基酸调控奶牛乳腺上皮细胞乳蛋白合成的研究进展

乳蛋白含量的高低是衡量乳品质的重要指标,奶牛乳腺上皮细胞（BMECs）是乳腺合成乳蛋白的基本功能单位。必需氨基酸（EAA）作为乳成分前体物质,其不仅是乳蛋白合成的底物,而且可以作为信号分子调控乳蛋白的合成。本文综述了近年关于EAA对BMECs乳蛋白合成的影响及其潜在的信号通路机制的研究进展,为今后系统研究氨基酸对BMECs乳蛋白合成的调控及提高泌乳奶牛氨基酸转化为乳蛋白的效率提供参考。     

基于AMPK信号通路研究LPS对奶牛乳腺上皮细胞脂代谢的调控机理

为了探讨脂多糖(LPS)对乳腺健康及乳脂合成的调控机制,本试验以奶牛乳腺上皮细胞系(MAC-T)为模型,通过向细胞中添加不同浓度的LPS (0 ng/mL、1 ng/mL、10 ng/mL、100 ng/mL、1000 ng/mL、10000 ng/mL),分别检测其细胞存活率、炎症因子、甘油三酯(TG)含量、腺苷酸活...     

金黄色葡萄球菌对奶牛乳腺上皮细胞Wnt/β-catenin信号通路和相关修复因子的影响

采用金黄色葡萄球菌侵袭原代奶牛乳腺上皮细胞,观察该病原菌对乳腺上皮细胞Wnt/β-catenin信号通路关键蛋白和相关修复因子EGFR、TGF-β3和VEGF基因mRNA表达的影响。金黄色葡萄球菌以MOI=1∶1的比例接种奶牛乳腺上皮细胞,分别作用0,15,30,45,60,120,240 min,采用Western blot法检测乳腺上皮细胞β-catenin、Cyclin D1及c-Myc蛋白表达水平;免疫荧光法检测β-catenin的表达及核易位;荧光定量PCR检测EGFR、TGF-β3和VEGF基因mRNA表达。结果显示,β-catenin蛋白在45,60,120 min表达量升高,与0 min相比差异显著(P<0.05);Cyclin D1蛋白在45,60,120和240 min表达量升高,差异显著或极显著(P<0.05或P<0.01);c-Myc蛋白在15,30,60,120,240 min表达量升高,差异极显著(P<0.01)。修复相关因子EGFR、TGF-β3和VEGF基因mRNA的表达量在30 min均出现极显著升高(P<0.01),且VEGF基因mRNA在45,60,120和240 min均呈现极显著升高(P<0.01),EGFR基因mRNA表达量在30,45和60 min时表达量极显著升高(P<0.01)。结果表明,金黄色葡萄球菌侵袭奶牛乳腺上皮细胞后,诱导Wnt/β-catenin信号通路的转导和修复因子EGFR、TGF-β3和VEGF基因转录,该信号通路和修复因子可能参与金黄色葡萄球菌导致的炎症和细胞损伤的修复过程。     

川芎嗪对LPS致伤奶牛子宫内膜上皮细胞的TLR4和BNBD4 mRNA表达的影响

为探讨脂多糖(LPS)对奶牛子宫内膜上皮细胞TLR4和BNBD4 m RNA表达的影响及川芎嗪的调控作用,体外培养奶牛子宫内膜上皮细胞,并分为对照组、LPS组、川芎嗪组(高、中、低浓度),利用实时荧光定量PCR法对各组细胞TLR4和BNBD4 m RNA表达进行了检测。结果表明:LPS可显著刺激奶牛子宫内膜上皮细胞TLR4及BNBD4 m RNA表达显著升高(P0.05),川芎嗪可抑制LPS诱导子宫内膜上皮细胞TLR4升高作用,并能促进BNBD4 m RNA表达。提示川芎嗪可干预LPS诱导的奶牛子宫内膜上皮细胞炎症反应,并促进其分泌β-防御素起到改善子宫内膜炎的作用。     

TLR4基因对奶牛乳房炎的影响研究

乳房炎一般是由乳房内感染引起的乳腺炎性疫病.目前,它是各国奶牛群中发生非常频繁的一种疫病.它可使奶牛产奶量和乳品质下降、泌乳持久力缩短和奶牛提早淘汰等,带来一定的经济损失.因此,降低奶牛乳房炎的发病率具有重要的经济意义.     

Cryopreserved bovine mammary cells to model epithelial response to infection

Mammary gland epithelial cells are likely to be important effectors in defending against mastitis, yet little is known about their response mechanisms. Here, we describe a cryopreserved bovine mammary epithelial cell model to study the infection response. Primary cell cultures from four Holstein cows were prepared, and frozen after two passages. The cell cultures from each cow were then thawed and maintained separately, yet simultaneously, and exposed to treatments that included infection with Staphylococcus aureus or exposure to LPS from Escherichia coli. A clear inflammatory response was shown by a significant (P < 0.05), dose dependent, increase of lactoferrin and IL-8 secretion within 24h in response to S. aureus or LPS. Marked increases (P < 0.05) in lactoferrin, TNF-alpha and serum amyloid A (SAA) mRNA expression were also observed. The results indicate the usefulness of our model to study infection responses of mammary epithelial cells, where all cells are simultaneously exposed to the same infection pressure. These responses can be studied over time, and most importantly, biological replication is provided by the four different genotypes being investigated individually. Finally, the results indicate that mammary epithelial cells play an important role in inflammatory response, through the production of pro-inflammatory cytokines, an acute phase protein, and lactoferrin.     

脂多糖诱导奶牛乳腺上皮细胞先天性免疫反应

采取荷斯坦奶牛乳腺,进行体外分离培养,并纯化细胞。用不同质量浓度(0、1、10、100mg/L)的脂多糖刺激乳腺上皮细胞,采用MTT法检测脂多糖对细胞增殖的影响,半定量PCR检测10mg/L的LPS对乳腺上皮细胞TLR4、TLR2、CD14、MD-2四个基因在不同时间(0、2、6h)mRNA表达水平的差异。结果表明,高剂量(100mg/L)的LPS对乳腺上皮细胞的增殖产生明显影响;LPS刺激乳腺上皮细胞后,导致TLR4、CD14、MD-2mRNA表达迅速升高,而TLR2mRNA弱表达。说明TLR4、CD14、MD-2参与LPS的识别,同时也说明脂多糖刺激乳腺上皮细胞后,乳腺上皮细胞能够产生先天性免疫反应。     

Deoxynivalenol induces oxidative stress,inflammatory response and apoptosis in bovine mammary epithelial cells

Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium graminearum. It is one of the most common feed contaminants that poses a serious threat to the health and performance of dairy cows. This study investigated the in vitro cytotoxicity of DON on bovine mammary epithelial cells (MAC‐T). DON at different concentrations (0.25, 0.3, 0.5, 0.8, 1 or 2 μg/ml) inhibited the growth of MAC‐T cells after 24 hr of exposure (p < .001). DON at 0.25 μg/ml increased lactate dehydrogenase (LDH) leakage (p < .05); decreased glutathione (GSH) levels (p < .001), total superoxide dismutase (T‐SOD) activity and total antioxidant capacity (T‐AOC; p < .01); and increased malondialdehyde (MDA) concentration (p < .01) in MAC‐T cells after 24 hr of exposure. We also observed that DON increased reactive oxygen species (ROS) levels in cells incubated for 9, 15 and 24 hr (p < .001). DON at 0.25 μg/ml triggered oxidative damage in MAC‐T cells. Furthermore, it induced an inflammatory response in the cells incubated for 9, 15 and 24 hr (p < .05) by increasing the mRNA expression levels of nuclear factor kappa B, myeloid differentiation factor 88 (MyD88), tumour necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, cyclooxygenase‐2 and IL‐8. We further examined the effect of DON on apoptosis. DON prevented normal proliferation of MAC‐T cells by blocked cell cycle progression in 24 hr (p < .001). In addition, the apoptosis rate measured using annexin V‐FITC significantly increased (p < .05) with increase in the mRNA expression level of Bax (p < .01) and increase in the Bax/Bcl‐2 ratio (p < .01) in cells incubated for 24 hr. In summary, DON exerts toxic effects in MAC‐T cells by causing oxidative stress, inducing an inflammatory response, affecting cell cycle and leading to apoptosis.     

牛乳腺上皮细胞的分离培养

为了对牛乳腺上皮细胞(MECs)进行分离、培养和鉴定,并研究细胞分泌功能,试验通过胶原酶消化法分离得到了牛乳腺上皮细胞,采用传代法对细胞进行纯化,对细胞标志蛋白进行免疫荧光染色鉴定,通过体外诱导和RT-PCR分析鉴定细胞的分泌功能。结果表明:分离到的牛乳腺上皮细胞具有典型乳腺上皮细胞的形态特征,表达广谱角蛋白,经诱导后可分泌β-酪蛋白。     

奶牛乳腺上皮细胞中Bta-miR-877靶基因的初步验证

利用高通量测序的方法筛选出在高脂奶牛和低脂奶牛中差异表达的miR-877和其预测的靶基因。通过生物信息学方法预测出edn1、ptgis可能是miR-877的靶基因,之后通过荧光定量检测miR-877以及其靶基因的表达量。结果显示,转染miR-877mimics组miR-877的相对表达量显著高于转染miR-877inhibitor组;靶基因的相对表达量,转染miR-877mimics组的edn1、ptgis基因表达水平均显著低于转染miR-877inhibitor组(P<0.01)。经GraphPad Prism6分析显示,edn1、ptgis基因的相对表达量与miR-877的表达量呈负相关,初步验证edn、ptgis是miR-877的靶基因。本试验为深入了解miR-877在乳脂质代谢调节中的作用提供依据,从而为生产实践提供一定的理论基础。