發(fā)布時(shí)間：2018-05-13 15:03

  本文選題：AWRK6 + 中和內(nèi)毒素　； 參考：《遼寧大學(xué)》2017年碩士論文

【摘要】：抗菌肽(Antibacterial peptides,AMPs)是一種生物體自身產(chǎn)生的小分子多肽,對(duì)細(xì)菌、病毒、原生生物和真菌都有抑制活性,對(duì)敗血癥等感染、炎癥性疾病有較好的治療作用。抗菌肽不僅可以使細(xì)胞膜通透性發(fā)生改變從而發(fā)揮殺菌作用,還可以作用于細(xì)胞內(nèi)部的其他靶標(biāo),介入細(xì)胞代謝活動(dòng),達(dá)到抑制和殺滅細(xì)菌的目的。與抗生素相比,抗菌肽殺菌時(shí)并不會(huì)造成內(nèi)毒素的釋放,也不會(huì)引起抗菌素耐藥性;在殺滅細(xì)菌的同時(shí)能中和內(nèi)毒素,表現(xiàn)出對(duì)內(nèi)毒素誘導(dǎo)的機(jī)體炎癥的抑制作用。AWRK6(SWVGKHGKKFGLKKHKKH)是根據(jù)東北林蛙皮膚抗菌肽Dybowskin-2CDYa,由本實(shí)驗(yàn)室優(yōu)化改造得到的新型抗菌肽,分子量為2130.5,是具有α-螺旋的陽(yáng)離子抗菌肽。前期研究表明,AWRK6具有良好的抑菌細(xì)菌活性,并且AWRK6在體內(nèi)外能顯著中和內(nèi)毒素,并能抑制由LPS所誘導(dǎo)的炎癥因子TNF-α與IL-8的釋放,AWRK6對(duì)小鼠腹腔巨噬細(xì)胞無(wú)毒性作用。AWRK6的這些優(yōu)點(diǎn),使其有可能成為一種新型的抗內(nèi)毒素藥物,用來治療內(nèi)毒素引發(fā)的敗血癥及敗血性休克。為進(jìn)一步研究AWRK6中和內(nèi)毒素機(jī)制,本文對(duì)AWRK6對(duì)LPS與內(nèi)毒素結(jié)合蛋白(lipopolysaccharide-binding protein,LBP)結(jié)合的影響及其對(duì)相關(guān)信號(hào)通路的mRNA表達(dá)水平與蛋白質(zhì)表達(dá)水平的影響進(jìn)行了研究。通過酶聯(lián)免疫吸附分析法,探究AWRK6對(duì)LPS與LBP結(jié)合的影響,以及對(duì)LPS-LBP復(fù)合物的影響,以初步研究其中和內(nèi)毒素的作用靶點(diǎn);使用昆明小鼠和小鼠腹腔巨噬細(xì)胞系,利用實(shí)時(shí)熒光定量PCR技術(shù)和蛋白質(zhì)免疫印跡技術(shù),研究了AWRK6對(duì)TLR4、JNK、IκB mRNA水平與TLR4、IκB蛋白表達(dá)水平以及IκB蛋白的磷酸化的影響,從而從而闡明AWRK6中和內(nèi)毒素的分子機(jī)制。研究結(jié)果表明,AWRK6對(duì)LPS與LBP的結(jié)合有顯著的抑制作用,并且對(duì)已經(jīng)聚合在一起的LPS-LBP復(fù)合體也有明顯的抑制作用;無(wú)論在體內(nèi)還是體外,AWRK6都對(duì)LPS誘導(dǎo)的TLR4、JNK mRNA表達(dá)量升高有顯著的抑制作用,對(duì)對(duì)LPS誘導(dǎo)的IκB mRNA表達(dá)量降低也有顯著的抑制作用,使其表達(dá)量基本恢復(fù)至正常的生理水平;AWRK6可以顯著抑制LPS誘導(dǎo)的TLR4、IκB蛋白表達(dá)量的變化與IκB蛋白的磷酸化,使其基本恢復(fù)至正常表達(dá)水平。本文初步研究闡明了AWRK6中和內(nèi)毒素的分子機(jī)制,為將AWRK6開發(fā)成為中和內(nèi)毒素的新型藥物奠定了堅(jiān)實(shí)的實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。
[Abstract]:Antimicrobial peptide Antibacterial peptide (AMPs) is a small molecule polypeptide produced by organism itself. It has inhibitory activity on bacteria, virus, protozoa and fungi, and has better therapeutic effect on septicemia and inflammatory diseases. Antimicrobial peptides can not only change the permeability of cell membrane to play a bactericidal effect, but also act on other targets within the cell, intervene in cell metabolism, and achieve the purpose of inhibiting and killing bacteria. Antimicrobial peptides do not cause endotoxin release and antimicrobial resistance compared with antibiotics; they neutralize endotoxin while killing bacteria. AWRK6 (SWVGKHGKKFGLKKHKKKH) is a new antimicrobial peptide, which was optimized by our laboratory and modified by our laboratory according to the skin antibacterial peptide Dybowskin-2 CDYa. its molecular weight is 2130.5, and it is a cationic antimicrobial peptide with 偽 -helix. Previous studies have shown that AWRK6 has good bacteriostasis activity, and AWRK6 can significantly neutralize endotoxin in vitro and in vivo, and inhibit the release of TNF- 偽 and IL-8 induced by LPS. AWRK6 has no toxic effect on murine peritoneal macrophages. It makes it possible to be a new anti-endotoxin drug in the treatment of sepsis and septic shock caused by endotoxin. In order to further study the mechanism of endotoxin neutralization by AWRK6, the effects of AWRK6 on the binding of LPS to lipopolysaccharide-binding protein (LBP) and the expression of mRNA and protein in related signaling pathway were studied. The effects of AWRK6 on the binding of LPS to LBP and on LPS-LBP complex were investigated by enzyme-linked immunosorbent assay (Elisa). The effects of AWRK6 on the expression of I 魏 B protein and the phosphorylation of I 魏 B protein in TLR4 were studied by using real-time fluorescence quantitative PCR and Western blot. The molecular mechanism of AWRK6 neutralizing endotoxin was elucidated. The results showed that AWRK6 could inhibit the binding of LPS to LBP and LPS-LBP complex which had been polymerized. Both in vivo and in vitro, AWRK6 could significantly inhibit the increase of LPS induced JNK mRNA expression in TLR4, as well as the decrease of I 魏 B mRNA expression induced by LPS. AWRK6 could significantly inhibit the changes of TLR4 I 魏 B protein expression and phosphorylation of I 魏 B protein induced by LPS, so that the expression level of I 魏 B protein returned to normal level. In this paper, the molecular mechanism of endotoxin neutralization by AWRK6 was preliminarily studied, which laid a solid experimental and theoretical basis for the development of AWRK6 as a new endotoxin neutralizing drug.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R91

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