本文選題：硒　切入點(diǎn)：金黃色葡萄球菌　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：硒在被發(fā)現(xiàn)的前期被認(rèn)為是一種有毒物質(zhì),但隨著研究的深入和科技的發(fā)展發(fā)現(xiàn)硒是多種生物體所必需的微量營(yíng)養(yǎng)物質(zhì),對(duì)人體及多種動(dòng)物器官的發(fā)育及生理功能的作用不可替代,硒缺乏會(huì)降低谷胱甘肽過(guò)氧化物酶的活性,進(jìn)而使自身的抗氧化能力減弱,體內(nèi)累積大量的過(guò)氧化物和脂質(zhì)自由基會(huì)破壞細(xì)胞和損傷組織,同時(shí)降低組織的免疫能力。硒補(bǔ)充與全身炎癥反應(yīng)綜合征和敗血癥的治愈有關(guān),體內(nèi)營(yíng)養(yǎng)硒攝入充分的個(gè)人,具有顯著的免疫刺激效應(yīng),可以增強(qiáng)活化和促進(jìn)增殖T細(xì)胞,增加NK細(xì)胞活性。乳腺炎是奶牛常發(fā)的乳腺組織炎癥性疾病,多發(fā)生于奶牛泌乳期。臨床上乳腺炎的主要致病菌是金黃色葡萄球菌,引發(fā)乳房紅腫,降低產(chǎn)奶量,嚴(yán)重感染時(shí)會(huì)導(dǎo)致奶牛全身性炎癥損傷。Micro RNA-155(miR-155)是一種典型的多生物學(xué)功能性miRNA分子,miR-155能影響造血細(xì)胞的分化,并在炎癥反應(yīng)、免疫反應(yīng)中發(fā)揮重要作用。炎癥刺激后miR-155在生物體內(nèi)表達(dá)上調(diào),還可被TLRs通過(guò)MyD88依賴性通路誘導(dǎo),后經(jīng)NF-κB和JNK/MAPK途徑調(diào)控炎癥反應(yīng)。但目前關(guān)于硒通過(guò)miR-155調(diào)控金葡菌誘導(dǎo)的細(xì)胞因子及TLR2表達(dá)進(jìn)而抑制NF-κB和MAPK的活化而減輕炎癥反應(yīng)的發(fā)生機(jī)制還尚不了解。本試驗(yàn)體內(nèi)實(shí)驗(yàn)部分通過(guò)飼喂SPF級(jí)BALB/c小鼠缺硒鼠糧、正常硒鼠糧、氋硒鼠糧60天,待雌鼠分娩5~7d后經(jīng)乳導(dǎo)管灌注金葡菌重懸液建立小鼠乳腺炎癥模型,研究硒對(duì)金葡菌性乳腺炎癥反應(yīng)的影響。經(jīng)乳腺組織硒含量檢測(cè)、乳腺病理組織學(xué)觀察、MPO活性檢測(cè)、miR-155及細(xì)胞因子和TLR1,2,6的表達(dá)分析,研究不同日糧硒水平對(duì)金葡菌誘導(dǎo)的小鼠乳腺炎癥反應(yīng)的影響。結(jié)果顯示,攝入不同日糧硒會(huì)影響乳腺組織硒含量,病理組織學(xué)分析表明金葡菌感染后缺硒小鼠乳腺有大量炎性細(xì)胞浸潤(rùn),腺泡結(jié)構(gòu)被破壞,而高硒小鼠乳腺結(jié)構(gòu)較完整。并且qPCR和ELISA檢測(cè)結(jié)果表明缺硒小鼠乳腺組織中miR-155和促炎因子表達(dá)水平顯著高于正常硒含量小鼠。這些結(jié)果說(shuō)明缺硒日糧促進(jìn)金葡菌誘導(dǎo)的小鼠乳腺炎癥反應(yīng),而補(bǔ)硒可以顯著抑制TLR2表達(dá),同時(shí)抑制其下游NF-κB和MAPK炎癥信號(hào)通路中蛋白磷酸化來(lái)降低促炎因子的產(chǎn)生,促進(jìn)乳腺組織的炎性修復(fù)功能。然后為了進(jìn)一步確證體內(nèi)實(shí)驗(yàn)結(jié)果,我們分離培養(yǎng)了原代小鼠乳腺上皮細(xì)胞(MMECs)并進(jìn)行體外實(shí)驗(yàn)。MMECs經(jīng)CK-18鑒定合格及MTT活性檢測(cè)后進(jìn)行miR-155-5p mimics轉(zhuǎn)染,然后用含不同硒濃度的Opti-MEM培養(yǎng)12h再添加金葡菌懸液構(gòu)建細(xì)胞模型,同時(shí)設(shè)立未轉(zhuǎn)染miR-155 mimics對(duì)照組。qPCR和ELISA檢測(cè)結(jié)果顯示轉(zhuǎn)染miR-155 mimics可以顯著增加促炎細(xì)胞因子和TLR2的表達(dá),增強(qiáng)NF-κB和MAPK蛋白磷酸化水平,表明miR-155在本試驗(yàn)中發(fā)揮促炎作用。但這一現(xiàn)象在實(shí)驗(yàn)組中隨著MSC中硒濃度增加被顯著抑制,并呈現(xiàn)劑量依賴關(guān)系,與在體動(dòng)物實(shí)驗(yàn)結(jié)果相一致。證實(shí)適量補(bǔ)充硒可以通過(guò)miR-155調(diào)控小鼠機(jī)體免疫功能,緩解乳腺炎癥損傷。
[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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.23

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本文編號(hào)：1559507