α-螺旋抗菌肽与传统抗生素的体内和体外的协同抗菌作用研究

发布时间：2018-11-13 09:36

【摘要】：众所周知，抗生素的泛滥使用导致耐药性的菌株不断出现，使得抗感染治疗愈加的困难和复杂化。传统的抗生素已经无法满足当前的治疗需求，而新型抗菌药物的研发速率也跟不上病原微生物引起的感染性疾病的出现速率。因此，越来越多的人在关注新型抗菌药物的研发，同时，也在积极探索新的治疗手段。抗菌肽（Antimicrobial peptides，，AMPs）是一类具有广谱抗菌活性的小分子多肽。随着研究的深入，人们发现抗菌肽在自然界中广泛存在，包括病毒、细菌、两栖类、昆虫、高等动植物乃至人体。抗菌肽（AMPs）对革兰阳性菌、革兰阴性菌和真菌均有抑杀作用，很多抗菌肽还可以抗原虫、抗病毒以及杀伤动物体内的肿瘤细胞，是一类极具潜力的多肽类抗生素。但是，抗菌肽在体内的不稳定性以及较高的毒副作用极大地限制了抗菌肽的临床应用。虽然抗菌肽作为候选药物应用于临床中还有一段距离，但是正在努力探索优化条件。目前，人们在探索新型治疗手段上已取得了一定的进展。抗生素的联合用药指的是，两种或者两种以上的抗生素同时应用于抗感染治疗中。联合用药可以扩大抗生素的抗菌范围，还可以减缓细菌耐药性的出现以及降低药物毒性的作用。在先前的研究中，人们利用抗生素之间的联合用药成功地提高了抗生素的治疗效力。但是，关于抗菌肽与传统抗生素之间的联合用药的研究还相对较少。本研究旨在探索抗菌肽与传统抗生素的联合用药，以期在提高其抗菌活性的同时降低多肽的毒副作用，并评估它们的疗效，探究它们之间的协同作用机制。首先，利用固相肽合成法，合成实验所需的6条多肽。同时，选取4种传统抗生素（亚胺培南，头孢吡肟，盐酸作用氟沙星和万古霉素），通过倍比稀释法分别测定抗菌肽与抗生素的抗菌活性，评估其体外疗效。数据表明，抗菌肽的最小抑菌浓度（Minimum inhibitory concentration, MIC）普遍小于传统抗生素，说明抗菌肽拥有更好的抗菌效力，这与先前的研究一致。其次，利用棋盘法测定抗菌肽与传统抗生素联合用药的协同抗菌效力。实验中选取了3种革兰氏阴性菌，3种革兰氏阳性菌。结果表明，抗菌肽与传统抗生素之间表现出较强的协同抗菌作用，没有任何拮抗作用的出现。最后，基于体外联合用药的结果，优选抗菌肽PL-5和盐酸左氧氟沙星作为实验用药，进行体内动物试验。通过创建小鼠皮肤感染模型，测定PL-5与盐酸左氧氟沙星之间的协同抗菌效力。研究结果显示，抗菌肽与盐酸左氧氟沙星表现出极强的体内协同抗菌效力。考虑到二者不同的杀菌机制，我们认为抗菌肽与传统抗生素之间的存在互补性的协同抗菌作用机制，从而提高了抗菌效力。通过该课题，我们可以确定，抗菌肽与传统抗生素之间的联合用药可以作为一个有效的疗法应用于未来的抗菌感染治疗中。
[Abstract]:It is well known that the prevalence of antibiotics leads to the emergence of drug-resistant strains, making anti-infection treatment more difficult and complicated. Traditional antibiotics can not meet the current treatment needs, and the rate of development of new antibiotics can not keep up with the emergence rate of infectious diseases caused by pathogenic microorganisms. Therefore, more and more people are paying attention to the research and development of new antimicrobial agents. Antimicrobial peptide (Antimicrobial peptides,AMPs) is a class of small peptides with broad spectrum antibacterial activity. With the development of research, antimicrobial peptides are widely found in nature, including viruses, bacteria, amphibians, insects, higher plants and animals and even human beings. Antimicrobial peptide (AMPs) can inhibit gram-positive bacteria, gram-negative bacteria and fungi. Many antimicrobial peptides can also inhibit antigens, antivirus and kill tumor cells in animals. However, the instability of antimicrobial peptides in vivo and high toxicity and side effects greatly limit the clinical application of antimicrobial peptides. Although antimicrobial peptides are still far from being used as candidates for clinical use, efforts are being made to explore optimal conditions. At present, people have made some progress in exploring new treatment methods. Combined use of antibiotics means that two or more antibiotics are used in anti-infective therapy at the same time. Combined use of antibiotics can expand the range of antibiotics, but also can reduce the emergence of bacterial drug resistance and reduce the toxicity of drugs. In previous studies, a combination of antibiotics was used to improve the efficacy of antibiotics. However, the research on the combination of antimicrobial peptides and traditional antibiotics is relatively rare. The purpose of this study was to explore the combination of antimicrobial peptides and traditional antibiotics in order to improve their antibacterial activity and reduce the toxic and side effects of peptides, evaluate their efficacy and explore the synergistic mechanism between them. Firstly, six polypeptides were synthesized by solid-phase peptide synthesis. Meanwhile, four traditional antibiotics (imipenem, cefepime, flufloxacin hydrochloric acid and vancomycin) were used to determine the antibacterial activity of antimicrobial peptides and antibiotics by double dilution method. The data showed that the minimum inhibitory concentration (Minimum inhibitory concentration, MIC) of antimicrobial peptides was generally lower than that of traditional antibiotics, which indicated that antimicrobial peptides had better antibacterial efficacy, which was consistent with previous studies. Secondly, chessboard method was used to determine the synergistic antibacterial efficacy of antimicrobial peptides combined with traditional antibiotics. Three Gram-negative bacteria and three Gram-positive bacteria were selected in the experiment. The results showed that there was a strong synergistic antibacterial effect between antimicrobial peptides and traditional antibiotics without any antagonism. Finally, the antibacterial peptide PL-5 and levofloxacin hydrochloride were selected as the experimental drugs. The synergistic antibacterial activity of PL-5 and levofloxacin hydrochloride was determined by establishing mouse skin infection model. The results showed that antimicrobial peptide and levofloxacin hydrochloride showed strong synergistic antibacterial effect in vivo. In view of their different bactericidal mechanisms, we think that there is a complementary synergistic antibacterial mechanism between antimicrobial peptides and traditional antibiotics, which improves the antibacterial efficacy. Through this study, we can confirm that the combination of antimicrobial peptides and traditional antibiotics can be used as an effective therapy for future antimicrobial infection treatment.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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