AWRK6中和内毒素的分子机制

发布时间：2018-05-13 15:03

本文选题：AWRK6 + 中和内毒素　；参考：《辽宁大学》2017年硕士论文

【摘要】：抗菌肽(Antibacterial peptides,AMPs)是一种生物体自身产生的小分子多肽,对细菌、病毒、原生生物和真菌都有抑制活性,对败血症等感染、炎症性疾病有较好的治疗作用。抗菌肽不仅可以使细胞膜通透性发生改变从而发挥杀菌作用,还可以作用于细胞内部的其他靶标,介入细胞代谢活动,达到抑制和杀灭细菌的目的。与抗生素相比,抗菌肽杀菌时并不会造成内毒素的释放,也不会引起抗菌素耐药性;在杀灭细菌的同时能中和内毒素,表现出对内毒素诱导的机体炎症的抑制作用。AWRK6(SWVGKHGKKFGLKKHKKH)是根据东北林蛙皮肤抗菌肽Dybowskin-2CDYa,由本实验室优化改造得到的新型抗菌肽,分子量为2130.5,是具有α-螺旋的阳离子抗菌肽。前期研究表明,AWRK6具有良好的抑菌细菌活性,并且AWRK6在体内外能显著中和内毒素,并能抑制由LPS所诱导的炎症因子TNF-α与IL-8的释放,AWRK6对小鼠腹腔巨噬细胞无毒性作用。AWRK6的这些优点,使其有可能成为一种新型的抗内毒素药物,用来治疗内毒素引发的败血症及败血性休克。为进一步研究AWRK6中和内毒素机制,本文对AWRK6对LPS与内毒素结合蛋白(lipopolysaccharide-binding protein,LBP)结合的影响及其对相关信号通路的mRNA表达水平与蛋白质表达水平的影响进行了研究。通过酶联免疫吸附分析法,探究AWRK6对LPS与LBP结合的影响,以及对LPS-LBP复合物的影响,以初步研究其中和内毒素的作用靶点;使用昆明小鼠和小鼠腹腔巨噬细胞系,利用实时荧光定量PCR技术和蛋白质免疫印迹技术,研究了AWRK6对TLR4、JNK、IκB mRNA水平与TLR4、IκB蛋白表达水平以及IκB蛋白的磷酸化的影响,从而从而阐明AWRK6中和内毒素的分子机制。研究结果表明,AWRK6对LPS与LBP的结合有显著的抑制作用,并且对已经聚合在一起的LPS-LBP复合体也有明显的抑制作用;无论在体内还是体外,AWRK6都对LPS诱导的TLR4、JNK mRNA表达量升高有显著的抑制作用,对对LPS诱导的IκB mRNA表达量降低也有显著的抑制作用,使其表达量基本恢复至正常的生理水平;AWRK6可以显著抑制LPS诱导的TLR4、IκB蛋白表达量的变化与IκB蛋白的磷酸化,使其基本恢复至正常表达水平。本文初步研究阐明了AWRK6中和内毒素的分子机制,为将AWRK6开发成为中和内毒素的新型药物奠定了坚实的实验基础和理论依据。
[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 伪 -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- 伪 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.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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