一种母乳来源的内源性多肽PDC213的抑菌活性与抑菌机制研究

发布时间：2018-05-20 10:52

本文选题：母乳 + 内源性多肽　；参考：《新乡医学院》2017年硕士论文

【摘要】：背景抗生素在目前临床医学中得到了广泛的应用,发挥了巨大的抗菌功能。但是由于抗生素不科学的应用,继而产生细菌耐药性,这可能制约着抗生素的应用与发展。目前研究发现,抗菌肽(antimicrobial peptides)因其独特的分子特性和抗菌活性,有望成为抗生素最为理想的替代药物。PDC213是从新生儿母乳中提取的一种内源性抗菌肽,具有较广泛的抑菌活性,将有良好的临床价值和应用前景。目的研究PDC213对新生儿重症监护病房(NICU)6种常见致病菌的抑菌活性与抑菌机制。方法(1).利用液相色谱串联质谱连用技术发现PDC213是一种在早产儿母乳中高表达的内源性多肽,通过生物信息学分析发现其是一种抑菌功能肽;(2)采用抑菌浓度曲线和抑菌圈实验分析PDC213的抑菌活性;(3)采用Live/Dead Bac Light TM bacterial viability kit染色结合荧光显微镜观察分析PDC213的抑菌活性与抑菌机制;(4)采用扫描电镜、透射电镜和DNA结合实验分析PDC213的抑菌机制。结果PDC213生物信息学分析显示PDC213是一种新型的抑菌肽。PDC213对NICU中常见致病菌金黄色葡萄球菌(S.aureus)和小肠结肠炎耶尔森氏菌(Y.enterocolitica)具有较强的抑菌活性,在一定时间内可有效快速杀死80%以上的S.aureus和Y.enterocolitica。抑菌圈实验结果显示S.aureus和Y.enterocolitica的抑菌圈平均直径分别为9.93 mm和9.83 mm。荧光显微镜显示S.aureus和Y.enterocolitica的镜下死亡细菌数实验组较对照组明显增多。扫描电镜和透射电镜实验显示经过PDC213刺激之后,S.aureus和Y.enterocolitica的表面形态和内部超微结构明显发生不规则改变,出现空泡等现象。DNA结合实验显示不同浓度的PDC213与一定量的核酸DNA结合后在1%琼脂糖凝胶上的显示条带无差异。结论PDC213对NICU中常见致病菌S.aureus和Y.enterocolitica具有较强的抑菌活性,PDC213是通过破坏S.aureus和Y.enterocolitica的细胞膜来杀死细菌,而不是通过和细胞内核酸结合来杀死细菌的。
[Abstract]:Background antibiotics are widely used in clinical medicine and play a great antibacterial function. However, the unscientific application of antibiotics leads to bacterial resistance, which may restrict the application and development of antibiotics. At present, it has been found that antimicrobial peptides. because of its unique molecular characteristics and antibacterial activity, it is expected to be the most ideal alternative to antibiotics. PDC213 is an endogenous antimicrobial peptide extracted from newborn breast milk and has a wide range of bacteriostatic activities. It will have good clinical value and application prospect. Objective to study the antimicrobial activity and mechanism of PDC213 against six common pathogenic bacteria in neonatal intensive care unit (NICU). Methods the method was used. Liquid chromatography-tandem mass spectrometry (LC-MS) showed that PDC213 was a highly expressed endogenous polypeptide in the breast milk of preterm infants. The bacteriostatic activity of PDC213 was analyzed by bioinformatics analysis, which was a kind of bacteriostasis peptide. (2) the bacteriostatic activity of PDC213 was analyzed by bacteriostatic concentration curve and bacteriostasis test. (3) the bacteriostatic activity of PDC213 was analyzed by Live/Dead Bac Light TM bacterial viability kit staining combined with fluorescence microscope. The mechanism of activity and bacteriostasis was characterized by scanning electron microscope (SEM). The bacteriostatic mechanism of PDC213 was analyzed by transmission electron microscope (TEM) and DNA. Results PDC213 bioinformatics analysis showed that PDC213 was a novel antimicrobial peptide. PDC213 had strong antibacterial activity against S.aureus and Y.enterocolitica, the common pathogenic bacteria in NICU. More than 80% of S.aureus and Y. enterocolitica could be killed quickly in a certain time. The results of bacteriostasis test showed that the average diameter of S.aureus and Y.enterocolitica were 9.93 mm and 9.83 mm respectively. Fluorescence microscope showed that the number of dead bacteria in S.aureus and Y.enterocolitica group was significantly higher than that in control group. Scanning electron microscopy and transmission electron microscopy showed that the surface morphology and internal ultrastructure of S.aureus and Y.enterocolitica changed irregularly after PDC213 stimulation. Vacuolation and other phenomena. DNA-binding experiments showed that there was no difference in the display bands between different concentrations of PDC213 and a certain amount of nucleic acid DNA on 1% agarose gel. Conclusion PDC213 has strong antibacterial activity against S.aureus and Y.enterocolitica in NICU. PDC213 kills bacteria by destroying the cell membrane of S.aureus and Y.enterocolitica, rather than killing bacteria by binding to intracellular nucleic acid.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R978

【参考文献】