【作者】 陈茹；

【导师】 栗震亚； 王锐；

【作者基本信息】 兰州大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 19世纪40年代,抗生素的发现引起了一场革命,从此人类对于感染性疾病不再束手无策。然而,由于抗生素在医药、农业及食品工业中的广泛应用及不合理使用,引起了耐药、泛耐药,交叉耐药,甚至是超级细菌的不断出现,使得抗感染问题变的越来越严峻,也使得人类的健康及生命安全面临极大威胁。因此,开发研究新的高效,低毒,具有新的抗菌作用机制的药物迫在眉睫。抗菌肽是生物体受到外界刺激时,发生免疫应答所诱导产生的具有强烈抗菌活性的物质,分子量在2000-7000左右,由20～60个氨基酸残基组成,具有广谱的抗真菌,抗细菌,抗病毒,抗肿瘤的活性。并且大量的研究表明抗菌肽主要是通过作用于病原体的细胞膜而发挥作用机制的,而一般的病原体很难通过改变自身的膜结构产生耐药性。因此,引起了人们的广泛关注。抗菌肽polybia-CP是从polybia paulista的毒液中提取出来的抗菌肽,序列为ILGTILGLLKSL-NH2,分子量为1239.73Da,是一种典型的两亲性阳离子多肽。在本研究中,我们用固相多肽合成方法合成了抗菌肽polybia-CP,用ESI-MS质谱确定它的分子量,并用HPLC分离纯化。接着我们以肿瘤细胞为模型,先研究了其对于生物细胞的毒性作用及作用模式。研究发现其主要通过破坏生物膜完整性而引起细胞死亡。用CD谱及计算机分子动力学模拟方法研究发现其在生物膜模拟环境中呈现a-螺旋结构,且该二级结构对于其发挥毒性至关重要。肿瘤细胞细胞表面分布有大量的负电荷,而阳离子的抗菌肽通常会作用于富含负电荷的肿瘤细胞表面。细菌细胞膜与肿瘤细胞膜具有一定的相似性,其表面也分布有大量负电荷。我们设想如果抗菌肽polybia-CP能够通过破坏细菌的细胞膜的方式发挥抗菌活性,那么这类药物可以规避传统抗生素的耐药机制的影响,而且细菌很难通过改变细胞膜的磷脂本质而发展对其的耐药性。因此,本研究通过多种手段重点研究了其对细菌细胞膜完整性及形态的影响；此外,为了排除细菌细胞膜上非磷脂成分对polybia-CP的作用影响,我们用磷脂(EYPC/EYPG 7：3)构建了模拟细菌细胞膜的磷脂泡(LUV),研究了polybia-CP对纯磷脂膜完整性的影响。研究结果表明,polybia-CP具有良好的抗菌活性,且其主要通过破坏细胞膜的完整性而发挥抗菌活性。本研究发现polybia-CP的膜破坏作用模式是不同于传统抗生素的一种新型作用模式,为开发对抗当前越来越严重的耐药现象的新型抗感染药物提供了新的策略,并具有很好的指导意义。更多还原

【Abstract】 In1940s, with the discovery of the antibiotics, there is a revolution in the medicine. Since then, people could cure a lot of infection diseases. However, as the extensive use of antibiotics in medicine, food and farming industry, the frequent emergency of resistant bacteria has brought great threat to life. The defeat between antibiotic and infection disease become more and more serious. All of this needs us to development new drugs with high activity, low mammalian toxicity and new antimicrobial activity exhibition modes. As their name implies, antimicrobial peptides (AMPs) are small molecules (usually from 2000 to 7000) with antimicrobial activity, and have 20 to 60 amino acids. Antimicrobial peptides (AMPs) constitute the first line of defense against invading microorganism. AMPs possess broad antimicrobial activity against bacteria, fungi, virus and tumor cells. The antimicrobial activity exhibition mode of AMPs has been associated with destruction of bacteria membrane and intracellular degradation events. It is now widely recognized that antimicrobial peptides (AMPs) could play a promising role in fighting the multi-drug resistant bacteria. Antimicrobial peptide polybia-CP was purified from the venom of the social wasp Polybia paulista. Its primary sequence was ILGTILGLLKSL-NH2(1239.73Da). In this study, we synthesized polybia-CP and use Electro Spray Ionization-Mass Spectroscopy to confirm its molecular weight. Reverse phase high-performance liquid chromatography was used to get purifier peptides. Because, there is similarities between the membrane of bacteria and tumor cells. At first, we studied the cytotoxicity and the exhibition mode of polybia-CP to tumor cells. In order to further characterize the mechanism of peptide-lipid bilayer of cell membranes, molecular dynamics (MD) simulation was employed to describe the spatial organization and temporal dynamics of the system. Our results revealed that polybia-CP has cytotoxicity to tumour cells and its site of action is the membrane of the tumor cells. Then, we studied the activity of polybia-CP to the bacteria including Gram-positive and Gram-negative bacteria. Furthermore, we use some methods to study the mechanism of polybia-CP to the bacteria. For example, in order to understand the influence of AMPs on the integrity of bacteria membrane, lipid membrane models, such as LUVs (EYPC/EYPG 7:3), was used in research work to exclude the influence of proteins and sugars. Our results found that polybia-CP has potent antimicrobial activity to both Gram-positive and Gram-negative bacteria. Both the real bacteria membrane and in vitro model membrane showed that polybia-CP is membrane active and its action target is the membrane of bacteria. It is difficult for bacteria to develop resistance to polybia-CP, which may offer a new strategy to defend the resistant bacteria in medicine, food and farming industry.So, our study will be useful for the development of new drugs.更多还原