人体防御素3对初期附着菌生物膜形成影响的实验研究

发布时间：2018-04-23 08:17

本文选题：人类防御素3(hBD3) + 链球菌属　；参考：《天津医科大学》2017年硕士论文

【摘要】：背景:人类疾病中,感染性疾病是导致生命死亡的主要疾病之一,其主要病原体是微生物。在现已发现的人类微生物感染性疾病中,研究发现有75%感染的发生与微生物生物膜形成有关,且微生物生物膜是造成口腔常见疾病的关键病因,例如:龋齿,牙周疾病,牙髓病,根尖周病和种植体周围炎等。根据世界卫生组织的调查显示,在众多工业化国家中,有60%-90%的学龄儿童和成人患龋率较高。此外,在全世界中有牙周病症状的成年人非常普遍,约有5%-20%的成人和2%的青少年患病成高发状态。研究表明,细菌是导致口腔生物膜疾病的首要原因。细菌生物膜的形成是一个多步骤且复杂的过程。随着初期附着菌的吸附,牙菌斑开始逐渐形成直至成熟,形成一个高度结构化且复杂的群体。有文献报道清洁牙面后的最初4小时内,粘附到牙齿表面的细菌有60%—90%为口链球菌(Streptococcus oralis,S.oralis),血链球菌(Streptococcus sanguinis,S.sanguinis),格登链球菌(Streptococcus gordonii,S.gordonii)。本研究采用人体β防御素(human beta-defensins hBDs)抑菌,根据现有研究基础及参考文献,为了最大限度的保留人体β防御素3(“human beta-defensins 3,hBD3”)的抑菌作用和生物稳定性,本课题合成hBD3的3条短肽hBD3-1,hBD3-2,hBD3-3,以抑制生物膜初期附着菌生长,进而抑制口腔生物膜的形成。本研究避免了抗菌肽刺激机体后产生的特异性免疫反应,防止了在高渗环境下抗菌肽失去抑菌活性,克服了药物抗菌所诱导的耐药性,为生成嵌合多肽,治疗相关感染性疾病开辟了新途径。目的:hBD3为“天然抗生素”中的一员,在口腔中作为第一道防御屏障,能够治疗革兰氏阳性菌(Gram-positive,G+)和革兰氏阴性菌(Gram-negative,G-)造成的感染性疾病。链球菌是牙菌斑形成过程中的初期附着菌,同时也是造成口腔牙菌斑疾病的重要原因。本论文设计合成hBD3的N端起始的三条短肽hBD3-1,hBD3-2,hBD3-3,评估hBD3-1,hBD3-2,hBD3-3对口腔链球菌的抑菌作用。方法:本研究中设计合成hBD3-1,hBD3-2,hBD3-3,检测hBD3-1,hBD3-2,hBD3-3对S.oralis,S.sanguinis和S.gordonii生物活性的抑制作用。通过最小抑菌浓度(Minimal Inhibitory Concentration MIC),最小杀菌浓度(Minimal Bactericidal Concentration MBC)和生物膜形成实验测试hBD3-1,hBD3-2,hBD3-3对不同状态下链球菌生长的抑菌作用。拉曼光谱(Raman spectroscopy)和圆二色光谱(Circular Dichroism spectroscopy)研究hBD3-1,hBD3-2,hBD3-3二级结构。通过激光共聚焦显微镜(Confocal Laser Scanning Microscopy CLSM)和电子扫描显微镜(Scanning Electron Microscopy SEM)观察链球菌细胞形态。结果:hBD3-1,hBD3-2,hBD3-3对链球菌的生长均具有抑制性,其中浮游状态下的S.oralis和S.sanguinis生物膜对hBD3-3的药物敏感性最强(P0.05)。Raman spectroscopy实验结果表明hBD3-1,hBD3-2,hBD3-3的二级结构中均具有酰胺Ⅰ和β折叠。但是在hBD3-2,hBD3-3的二级结构中未发现酪氨酸。CD spectroscopy实验结果表明在口腔环境中hBD3-1,hBD3-2,hBD3-3抑菌活性保持稳定,不会因为温度的变化影响抑菌作用,且不受二硫键的影响。通过CLSM镜下观察染色的链球菌生物膜发现作用于hBD3-3的细菌生物膜的凋亡细胞染色比例要多于对照组。SEM镜下观察到hBD3-3对链球菌生物膜生物量的减少和细菌形态的破坏。结论:hBD3-3作为hBD3 45氨基酸序列中的一段短肽,能够有效抑制初期附着菌的生长,进而抑制口腔生物膜的形成,最终预防和降低口腔生物膜感染性疾病,维持口腔健康。
[Abstract]:Background: in human diseases, infectious diseases are one of the major diseases leading to life death. The main pathogen is microorganism. In the human microbial infectious diseases discovered now, 75% infections are found to be related to the formation of microbial biofilm, and the microbial biofilm is the key cause of common oral diseases. Such as dental caries, periodontitis, dental pulp disease, periapical disease and periimplant inflammation. According to the WHO survey, the prevalence rate of 60%-90% for school-age children and adults is high in many industrialized countries. In addition, adults with periodontitis symptoms in the world are very common, about 5%-20% adults and 2% of adolescents are ill. The formation of bacteria is the leading cause of the oral biofilm disease. The formation of bacterial biofilms is a multistep and complex process. With the initial attachment of the bacteria, the plaque begins to form and matures, forming a highly structured and complex group. The first 4 after cleaning the tooth surface has been reported. Within hours, bacteria adhered to the surface of the teeth were 60% - 90% for Streptococcus oralis (S.oralis), Streptococcus (Streptococcus sanguinis, S.sanguinis), and Streptococcus Gdon (Streptococcus gordonii, S.gordonii). This study used human beta defensin (human beta-defensins hBDs) to bacteriostasis, based on existing research basis and reference. In order to maximize the bacteriostasis and biological stability of human beta defensin 3 ("human beta-defensins 3, hBD3"), 3 short peptides of hBD3, hBD3-1, hBD3-2, hBD3-3, were synthesized to inhibit the growth of the early adherent bacteria in the biofilm and inhibit the formation of the oral biofilm. This study avoided the antiseptic peptide stimulation of the body. The specific immune response prevents the antibacterial peptide from losing its antibacterial activity in the hypertonic environment and overcomes the drug resistance induced by the drug. It opens a new way for the generation of chimeric peptides and the treatment of related infectious diseases. Objective: hBD3 is a member of "natural antibiotics", as the first defense barrier in the mouth, and can be used to treat gram blue. Infectious diseases caused by Gram-positive (G+) and Gram-negative bacteria (Gram-negative, G-). Streptococcus is an initial attachment bacteria in the formation of dental plaque, and is also an important cause of dental plaque disease. This paper designs and syntheses three short peptide hBD3-1, hBD3-2, hBD3-3, hBD3-1, hBD3-2, h in the N terminal of hBD3. The inhibitory effect of BD3-3 on oral Streptococcus. Methods: hBD3-1, hBD3-2, and hBD3-3 were designed and synthesized in this study. The inhibitory effects of hBD3-1, hBD3-2, hBD3-3 on the biological activity of S.oralis, S.sanguinis and S.gordonii were investigated. BC) and biofilm formation experiment test the Bacteriostasis of hBD3-1, hBD3-2, hBD3-3 on the growth of Streptococcus in different states. Raman spectroscopy (Raman spectroscopy) and circular two color spectra (Circular Dichroism spectroscopy) study hBD3-1, hBD3-2, hBD3-3 two structure. The morphology of streptococcal cells was observed by Scanning Electron Microscopy SEM. Results: hBD3-1, hBD3-2, hBD3-3 had inhibitory effects on the growth of Streptococcus, and the S.oralis and S.sanguinis biofilms under the planktonic state were the most sensitive to hBD3-3 (P0.05). Amides I and beta folds were all in the two structure of 3-3. However, no tyrosine.CD spectroscopy experimental results were found in the two grade structure of hBD3-2, hBD3-3 indicating that the Bacteriostasis of hBD3-1, hBD3-2, hBD3-3 remained stable in the oral environment, and did not affect the bacteriostasis because of the change of temperature, and was not affected by the two sulfur bond. The coloring streptococcal biofilm found that the proportion of apoptotic cells in the bacterial biofilm of hBD3-3 was more than that of the control group under the.SEM microscope to observe the decrease of the biofilm biomass of Streptococcus and the destruction of bacteria morphology. Conclusion: as a short peptide in the sequence of hBD3 45 amino acids, hBD3-3 can effectively inhibit the initial attachment bacteria. Growth, thereby inhibiting the formation of oral biofilm, and ultimately prevent and reduce oral biofilm infectious diseases, and maintain oral health.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R780.2

【参考文献】