TIR结构域来源封闭肽对LPS诱导小鼠乳腺炎的保护作用机制

发布时间：2018-01-06 21:08

本文关键词：TIR结构域来源封闭肽对LPS诱导小鼠乳腺炎的保护作用机制　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：奶牛乳腺炎是造成奶牛养殖业重大经济损失的主要疾病之一,大肠杆菌等革兰氏阴性菌感染乳腺组织导致的剧烈炎症反应引起的免疫损伤是奶牛乳腺炎发生的主要原因。脂多糖(LPS)是大肠杆菌诱发机体剧烈炎症反应的主要毒力因子,乳导管单独灌注LPS可引起与大肠杆菌感染完全相同的临床症状。研究发现LPS引起的剧烈炎症反应是由宿主天然免疫系统中的TLR4识别并向下游转导信号,引起相关通路终点核转录因子入核,控制细胞因子基因表达的结果。TLR4及其下游衔接分子都存在TIR结构域,衔接蛋白分子通过彼此之间的TIR结构域相互作用,保证通路蛋白分子正常衔接,使得信号通路正常转导,本课题通过模拟衔接分子TIR结构域的空间构象合成可干扰TIR-TIR相互作用的封闭肽,借以阻断TLR4信号通路的正常转导,用于控制机体的炎症反应。本实验将已经合成并且筛选出来的具有抗炎作用的封闭肽TM6和TR6用于研究其对LPS诱导的小鼠乳腺炎的保护作用及保护机制。首先通过LPS乳导管灌注的方法建立小鼠乳腺炎动物模型,并且分别给予不同浓度的预先筛选出的具有抗炎作用的封闭肽TM6或TR6。LPS刺激24小时之后收集乳腺组织样品,通过HE方法检测乳腺组织的病理学变化;通过ELISA方法检测乳腺组织炎性细胞因子TNF-α、IL-1β、IL-6的表达情况;通过MPO试剂盒检测乳腺组织MPO活性变化评价TM6或TR6对LPS诱导的小鼠乳腺炎的保护作用。实验结果表明,与对照组相比,LPS刺激的小鼠乳腺组织发生明显的病理学变化,如腺泡形态发生改变、炎性细胞因子浸润、充血等现象,此外乳腺组织炎性细胞因子TNF-α、IL-1β、IL-6和MPO活性也明显升高,然而不同剂量的TM6或TR6抑制了LPS诱导的乳腺组织炎性变化,并且呈剂量依赖性,以上的结果表明,在LPS诱导的小鼠乳腺炎动物模型中,封闭肽TM6或TR6对乳腺炎症具有保护作用。为了进一步研究封闭肽对LPS诱导的小鼠乳腺炎保护作用的机制,我们通过酶消化法提取小鼠的乳腺上皮细胞用于下一步实验。首先通过MTT法检测TM6或TR6在所用剂量0-20μM范围内对乳腺上皮细胞没有细胞毒性作用,之后以1μg/ml的LPS刺激乳腺上皮细胞,并且用不同浓度的TM6或TR6预处理1小时,收集样品,通过ELISA方法检测炎性细胞因子的表达;通过western blot方法检测NF-κB和MAPK信号通路的表达。实验结果表明,不同浓度的TM6或TR6明显的抑制了LPS刺激小鼠乳腺上皮细胞引起的炎性细胞因子TNF-α和IL-6的表达,并且TM6或TR6能够进一步抑制NF-κB和MAPK信号通路相关蛋白的表达。以上实验结果表明,封闭肽TM6或TR6可能是通过抑制NF-κB和MAPK信号通路的活化进而抑制炎性细胞因子的表达,从而对乳腺炎起到保护性的作用。综上所述,本研究首先通过LPS诱导的小鼠乳腺炎动物模型证明封闭肽TM6或TR6对乳腺组织的损伤具有保护性作用,为了进一步研究TM6或TR6对乳腺组织损伤的保护作用机制,我们通过体外培养的乳腺上皮细胞为实验工具,进一步表明了封闭肽TM6或TR6可能是通过抑制LPS诱导的NF-κB和MAPK信号通路的活化抑制炎性细胞因子的表达,进而对LPS诱导的乳腺炎起到保护作用。这些结果表明针对先天性免疫信号通路TLR4及相关衔接蛋白合成不同的封闭肽抑制炎性信号的正常转导,可能成为未来新抗炎制剂开发的方向。
[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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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