硒通过microRNA 155调控金黄色葡萄球菌性乳腺炎发生的作用机制研究

发布时间：2018-03-03 04:20

本文选题：硒　切入点：金黄色葡萄球菌　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：硒在被发现的前期被认为是一种有毒物质,但随着研究的深入和科技的发展发现硒是多种生物体所必需的微量营养物质,对人体及多种动物器官的发育及生理功能的作用不可替代,硒缺乏会降低谷胱甘肽过氧化物酶的活性,进而使自身的抗氧化能力减弱,体内累积大量的过氧化物和脂质自由基会破坏细胞和损伤组织,同时降低组织的免疫能力。硒补充与全身炎症反应综合征和败血症的治愈有关,体内营养硒摄入充分的个人,具有显著的免疫刺激效应,可以增强活化和促进增殖T细胞,增加NK细胞活性。乳腺炎是奶牛常发的乳腺组织炎症性疾病,多发生于奶牛泌乳期。临床上乳腺炎的主要致病菌是金黄色葡萄球菌,引发乳房红肿,降低产奶量,严重感染时会导致奶牛全身性炎症损伤。Micro RNA-155(miR-155)是一种典型的多生物学功能性miRNA分子,miR-155能影响造血细胞的分化,并在炎症反应、免疫反应中发挥重要作用。炎症刺激后miR-155在生物体内表达上调,还可被TLRs通过MyD88依赖性通路诱导,后经NF-κB和JNK/MAPK途径调控炎症反应。但目前关于硒通过miR-155调控金葡菌诱导的细胞因子及TLR2表达进而抑制NF-κB和MAPK的活化而减轻炎症反应的发生机制还尚不了解。本试验体内实验部分通过饲喂SPF级BALB/c小鼠缺硒鼠粮、正常硒鼠粮、氋硒鼠粮60天,待雌鼠分娩5~7d后经乳导管灌注金葡菌重悬液建立小鼠乳腺炎症模型,研究硒对金葡菌性乳腺炎症反应的影响。经乳腺组织硒含量检测、乳腺病理组织学观察、MPO活性检测、miR-155及细胞因子和TLR1,2,6的表达分析,研究不同日粮硒水平对金葡菌诱导的小鼠乳腺炎症反应的影响。结果显示,摄入不同日粮硒会影响乳腺组织硒含量,病理组织学分析表明金葡菌感染后缺硒小鼠乳腺有大量炎性细胞浸润,腺泡结构被破坏,而高硒小鼠乳腺结构较完整。并且qPCR和ELISA检测结果表明缺硒小鼠乳腺组织中miR-155和促炎因子表达水平显著高于正常硒含量小鼠。这些结果说明缺硒日粮促进金葡菌诱导的小鼠乳腺炎症反应,而补硒可以显著抑制TLR2表达,同时抑制其下游NF-κB和MAPK炎症信号通路中蛋白磷酸化来降低促炎因子的产生,促进乳腺组织的炎性修复功能。然后为了进一步确证体内实验结果,我们分离培养了原代小鼠乳腺上皮细胞(MMECs)并进行体外实验。MMECs经CK-18鉴定合格及MTT活性检测后进行miR-155-5p mimics转染,然后用含不同硒浓度的Opti-MEM培养12h再添加金葡菌悬液构建细胞模型,同时设立未转染miR-155 mimics对照组。qPCR和ELISA检测结果显示转染miR-155 mimics可以显著增加促炎细胞因子和TLR2的表达,增强NF-κB和MAPK蛋白磷酸化水平,表明miR-155在本试验中发挥促炎作用。但这一现象在实验组中随着MSC中硒浓度增加被显著抑制,并呈现剂量依赖关系,与在体动物实验结果相一致。证实适量补充硒可以通过miR-155调控小鼠机体免疫功能,缓解乳腺炎症损伤。
[Abstract]:In the early stage of selenium was found to be considered a toxic substance, but with the development of research and technology found that selenium is the essential nutrients in various organisms, developmental and physiological functions of the human body and a variety of animal organs plays an irreplaceable role, will reduce the glutathione peroxidase activity of selenium deficiency, and then make the the antioxidant capacity decreased, accumulation of a large number of lipid peroxides and free radicals can damage cells and tissue damage, and reduce tissue immunity. Selenium supplement and systemic inflammatory response syndrome and sepsis related comprehensive cure, nutritional selenium intake is sufficient, with significant immune stimulating effect, can enhance the activation and proliferation T cells, increase the activity of NK cells. The breast tissue inflammatory disease mastitis is often made of cow and cow occurred in the lactation period. The main clinical breast inflammation To bacterial pathogens are Staphylococcus aureus, causing breast swelling, reduced milk yield, severe infection can lead to systemic inflammatory injury in.Micro cows (miR-155 RNA-155) is a typical multi biological functional miRNA molecules, miR-155 can interfere the differentiation of hematopoietic cells, and play an important role in inflammation, immune response after the stimulation of inflammation. MiR-155 in vivo expression can also be TLRs via a MyD88 dependent pathway induced by NF- kappa B and JNK/MAPK pathways regulate inflammation. But on the selenium by regulating miR-155 cytokines and TLR2 expression induced by S.aureus activation and inhibit NF- kappa B and MAPK occurrence mechanism inflammatory reaction is still not understood. This experiment in vivo in selenium deficient rat food by feeding SPF BALB/c mice, normal rat food selenium, selenium Meng rat food for 60 days, the female rats 5~7d after delivery catheter infusion of Staphylococcus aureus in milk Bacteria re suspension mice breast inflammation model, effects of selenium on Staphylococcus aureus of breast inflammation. The detection of selenium content in breast tissue, breast histological observation and MPO activity detection, analysis and expression of miR-155 cytokines and TLR1,2,6, effects of different dietary selenium levels on mouse mammary inflammatory response induced by Staphylococcus aureus bacteria. The results showed that different dietary selenium intake will affect the selenium content of breast tissue, histological analysis showed that Staphylococcus aureus infection after SE deficient mice breast inflammatory cell infiltration, alveolar structure was damaged, and the high selenium mouse mammary structure is complete. And the qPCR and ELISA results showed that miR-155 of selenium deficiency mouse mammary gland tissue and proinflammatory cytokine expression level was significantly higher than that in normal mice. These results indicated that the content of selenium in selenium deficient diets promote mouse breast inflammation induced by Staphylococcus aureus, and selenium can significantly Inhibition of TLR2 expression, and inhibition of protein phosphorylation of downstream NF- kappa B and MAPK in the inflammatory signaling pathway to reduce the production of pro-inflammatory cytokines, promote the inflammatory repair function of breast tissue. Then in order to further confirm the in vivo experiment results, we isolated and cultured primary mouse mammary epithelial cells (MMECs) and in vitro by.MMECs the detection of CK-18 accreditation and the activity of MTT after miR-155-5p mimics transfection, and then add the Staphylococcus aureus suspension and then constructed a cell model with different concentration of Se Opti-MEM 12h culture, while the establishment of untransfected miR-155 mimics control group.QPCR and ELISA showed that miR-155 mimics transfection can significantly increase the expression of proinflammatory cytokines and TLR2, enhanced the expression of NF- B and phosphorylation of MAPK protein, showed that miR-155 in this experiment play proinflammatory effects. But this phenomenon in the experimental group with MSC was significantly increased in selenium concentration Inhibition and dose-dependent relationship were consistent with the in vivo animal experiment. It was confirmed that proper supplementation of selenium can regulate the immune function of mice through miR-155, and alleviate the inflammatory injury of mammary gland.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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