本文關(guān)鍵詞：TIR結(jié)構(gòu)域來(lái)源封閉肽對(duì)LPS誘導(dǎo)小鼠乳腺炎的保護(hù)作用機(jī)制　出處：《吉林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乳腺炎 脂多糖 封閉肽 細(xì)胞因子 NF-κB MAPK

【摘要】：奶牛乳腺炎是造成奶牛養(yǎng)殖業(yè)重大經(jīng)濟(jì)損失的主要疾病之一,大腸桿菌等革蘭氏陰性菌感染乳腺組織導(dǎo)致的劇烈炎癥反應(yīng)引起的免疫損傷是奶牛乳腺炎發(fā)生的主要原因。脂多糖(LPS)是大腸桿菌誘發(fā)機(jī)體劇烈炎癥反應(yīng)的主要毒力因子,乳導(dǎo)管單獨(dú)灌注LPS可引起與大腸桿菌感染完全相同的臨床癥狀。研究發(fā)現(xiàn)LPS引起的劇烈炎癥反應(yīng)是由宿主天然免疫系統(tǒng)中的TLR4識(shí)別并向下游轉(zhuǎn)導(dǎo)信號(hào),引起相關(guān)通路終點(diǎn)核轉(zhuǎn)錄因子入核,控制細(xì)胞因子基因表達(dá)的結(jié)果。TLR4及其下游銜接分子都存在TIR結(jié)構(gòu)域,銜接蛋白分子通過(guò)彼此之間的TIR結(jié)構(gòu)域相互作用,保證通路蛋白分子正常銜接,使得信號(hào)通路正常轉(zhuǎn)導(dǎo),本課題通過(guò)模擬銜接分子TIR結(jié)構(gòu)域的空間構(gòu)象合成可干擾TIR-TIR相互作用的封閉肽,借以阻斷TLR4信號(hào)通路的正常轉(zhuǎn)導(dǎo),用于控制機(jī)體的炎癥反應(yīng)。本實(shí)驗(yàn)將已經(jīng)合成并且篩選出來(lái)的具有抗炎作用的封閉肽TM6和TR6用于研究其對(duì)LPS誘導(dǎo)的小鼠乳腺炎的保護(hù)作用及保護(hù)機(jī)制。首先通過(guò)LPS乳導(dǎo)管灌注的方法建立小鼠乳腺炎動(dòng)物模型,并且分別給予不同濃度的預(yù)先篩選出的具有抗炎作用的封閉肽TM6或TR6。LPS刺激24小時(shí)之后收集乳腺組織樣品,通過(guò)HE方法檢測(cè)乳腺組織的病理學(xué)變化;通過(guò)ELISA方法檢測(cè)乳腺組織炎性細(xì)胞因子TNF-α、IL-1β、IL-6的表達(dá)情況;通過(guò)MPO試劑盒檢測(cè)乳腺組織MPO活性變化評(píng)價(jià)TM6或TR6對(duì)LPS誘導(dǎo)的小鼠乳腺炎的保護(hù)作用。實(shí)驗(yàn)結(jié)果表明,與對(duì)照組相比,LPS刺激的小鼠乳腺組織發(fā)生明顯的病理學(xué)變化,如腺泡形態(tài)發(fā)生改變、炎性細(xì)胞因子浸潤(rùn)、充血等現(xiàn)象,此外乳腺組織炎性細(xì)胞因子TNF-α、IL-1β、IL-6和MPO活性也明顯升高,然而不同劑量的TM6或TR6抑制了LPS誘導(dǎo)的乳腺組織炎性變化,并且呈劑量依賴性,以上的結(jié)果表明,在LPS誘導(dǎo)的小鼠乳腺炎動(dòng)物模型中,封閉肽TM6或TR6對(duì)乳腺炎癥具有保護(hù)作用。為了進(jìn)一步研究封閉肽對(duì)LPS誘導(dǎo)的小鼠乳腺炎保護(hù)作用的機(jī)制,我們通過(guò)酶消化法提取小鼠的乳腺上皮細(xì)胞用于下一步實(shí)驗(yàn)。首先通過(guò)MTT法檢測(cè)TM6或TR6在所用劑量0-20μM范圍內(nèi)對(duì)乳腺上皮細(xì)胞沒(méi)有細(xì)胞毒性作用,之后以1μg/ml的LPS刺激乳腺上皮細(xì)胞,并且用不同濃度的TM6或TR6預(yù)處理1小時(shí),收集樣品,通過(guò)ELISA方法檢測(cè)炎性細(xì)胞因子的表達(dá);通過(guò)western blot方法檢測(cè)NF-κB和MAPK信號(hào)通路的表達(dá)。實(shí)驗(yàn)結(jié)果表明,不同濃度的TM6或TR6明顯的抑制了LPS刺激小鼠乳腺上皮細(xì)胞引起的炎性細(xì)胞因子TNF-α和IL-6的表達(dá),并且TM6或TR6能夠進(jìn)一步抑制NF-κB和MAPK信號(hào)通路相關(guān)蛋白的表達(dá)。以上實(shí)驗(yàn)結(jié)果表明,封閉肽TM6或TR6可能是通過(guò)抑制NF-κB和MAPK信號(hào)通路的活化進(jìn)而抑制炎性細(xì)胞因子的表達(dá),從而對(duì)乳腺炎起到保護(hù)性的作用。綜上所述,本研究首先通過(guò)LPS誘導(dǎo)的小鼠乳腺炎動(dòng)物模型證明封閉肽TM6或TR6對(duì)乳腺組織的損傷具有保護(hù)性作用,為了進(jìn)一步研究TM6或TR6對(duì)乳腺組織損傷的保護(hù)作用機(jī)制,我們通過(guò)體外培養(yǎng)的乳腺上皮細(xì)胞為實(shí)驗(yàn)工具,進(jìn)一步表明了封閉肽TM6或TR6可能是通過(guò)抑制LPS誘導(dǎo)的NF-κB和MAPK信號(hào)通路的活化抑制炎性細(xì)胞因子的表達(dá),進(jìn)而對(duì)LPS誘導(dǎo)的乳腺炎起到保護(hù)作用。這些結(jié)果表明針對(duì)先天性免疫信號(hào)通路TLR4及相關(guān)銜接蛋白合成不同的封閉肽抑制炎性信號(hào)的正常轉(zhuǎn)導(dǎo),可能成為未來(lái)新抗炎制劑開(kāi)發(fā)的方向。
[Abstract]:Cow mastitis is one of the main diseases that cause significant economic losses in dairy industry, immune injury caused severe inflammation of breast tissue caused by Escherichia coli and other gram negative bacteria infection is a major cause of mastitis occurrence. Lipopolysaccharide (LPS) is the major virulence factors of Escherichia coli induce severe inflammation, the milk ducts can cause LPS perfusion alone the infection of Escherichia coli and the clinical symptoms are exactly the same. The study found that severe inflammatory reaction caused by LPS was identified by TLR4 in the natural immune system of the host and downstream signal transduction pathway, leading end point of nuclear transcription factor in the nucleus, control of cytokine gene expression results of.TLR4 and its downstream adaptor molecule are TIR domain, cohesion protein molecules through TIR domain interactions between protein molecules, which makes the normal pathway connection, signal pathway Normal transduction, through the simulation of space conformation adaptor molecule TIR domain synthesis blocking peptide interference TIR-TIR interaction, so as to block the normal transduction of the TLR4 signaling pathway for inflammation control body. This experiment will have been synthesized and screened with the anti-inflammatory effect of TM6 and TR6 for blocking peptide on mice mastitis induced by LPS and protective effect of the protection mechanism. Firstly, by means of LPS catheter to establish mouse mastitis dairy animal model, and given different concentration respectively in advance after the antiinflammatory effects of blocking peptide TM6 or TR6.LPS stimulation screened 24 hours to collect breast tissue samples, the changes of pathology in breast tissue by detecting method of HE; through the ELISA method for detection of breast tissue inflammatory cytokines TNF- alpha, IL-1 beta, IL-6 expression; through the breast tissue MPO kit to detect MPO The protective effect of the activity change of evaluation of TM6 or TR6 on mouse mastitis induced by LPS. The experimental results show that compared with the control group, LPS stimulated mouse mammary tissue had obvious pathological changes, such as acinar morphological changes, infiltration of inflammatory cytokines, congestion and other phenomena, in addition to breast tissue inflammatory cell factor TNF- alpha. IL-1 IL-6 beta, and MPO activity was significantly increased, however, different doses of TM6 or TR6 inhibited breast tissue LPS induced inflammatory changes in a dose-dependent manner, the above results show that the animal model of LPS induced mouse mastitis, blocking peptide TM6 or TR6 has a protective effect on mastitis. In order to further study on the mechanism of the protective effect of blocking peptide LPS induced mouse mastitis, we through enzyme digestion extraction of mouse mammary epithelial cells for the next experiment. First detected by MTT or TR6 in the TM6 The dose of 0-20 M in the range of breast epithelial cells without cytotoxicity, followed by 1 g/ml LPS stimulation of mammary epithelial cells, and with different concentrations of TM6 or TR6 pretreatment for 1 hours, collect the sample, through the expression of inflammatory cytokines ELISA expression by Western detection method; blot method for detection of B and NF- k the MAPK signaling pathway. The experimental results show that the expression of different concentrations of TM6 or TR6 significantly inhibited LPS induced mouse mammary epithelial cells inflammatory cytokines TNF- and IL-6, and the TM6 or TR6 can express further inhibition of NF- K B and MAPK signaling pathway related proteins. These results indicate that blocking peptide TM6 or TR6 may be expressed by suppressing activation of NF- kappa B and MAPK signaling pathway and inhibition of inflammatory cytokines, which play a protective role for mastitis. In summary, this study first induced by LPS Animal model of mastitis mice that blocking peptide TM6 or TR6 has protective effect on breast tissue damage to the protective mechanism of breast tissue injury in the further study of TM6 or TR6, we cultured mammary epithelial cells as experimental tools to further show that the closed peptide TM6 or TR6 may be activated by inhibiting the expression of inflammatory cells the inhibition of LPS induced NF- factor kappa B and MAPK signaling pathways, and protective effects on Mastitis Induced by LPS. These results indicate that the normal transduction of innate immune signaling pathways linking TLR4 and related protein synthesis blocking peptide to inhibit inflammatory signals, may become the future direction of the development of new anti-inflammatory agents.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.23

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Guo-Ling Chen;Jing-Jing Zhang;Xin Kao;Lu-Wan Wei;Zhi-Yu Liu;;Emodin ameliorates lipopolysaccharides-induced corneal inflammation in rats[J];International Journal of Ophthalmology;2015年04期

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本文編號(hào)：1389519