本文選題：人類(lèi)防御素3(hBD3) + 鏈球菌屬　； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:人類(lèi)疾病中,感染性疾病是導(dǎo)致生命死亡的主要疾病之一,其主要病原體是微生物。在現(xiàn)已發(fā)現(xiàn)的人類(lèi)微生物感染性疾病中,研究發(fā)現(xiàn)有75%感染的發(fā)生與微生物生物膜形成有關(guān),且微生物生物膜是造成口腔常見(jiàn)疾病的關(guān)鍵病因,例如:齲齒,牙周疾病,牙髓病,根尖周病和種植體周?chē)椎�。根�?jù)世界衛(wèi)生組織的調(diào)查顯示,在眾多工業(yè)化國(guó)家中,有60%-90%的學(xué)齡兒童和成人患齲率較高。此外,在全世界中有牙周病癥狀的成年人非常普遍,約有5%-20%的成人和2%的青少年患病成高發(fā)狀態(tài)。研究表明,細(xì)菌是導(dǎo)致口腔生物膜疾病的首要原因。細(xì)菌生物膜的形成是一個(gè)多步驟且復(fù)雜的過(guò)程。隨著初期附著菌的吸附,牙菌斑開(kāi)始逐漸形成直至成熟,形成一個(gè)高度結(jié)構(gòu)化且復(fù)雜的群體。有文獻(xiàn)報(bào)道清潔牙面后的最初4小時(shí)內(nèi),粘附到牙齒表面的細(xì)菌有60%—90%為口鏈球菌(Streptococcus oralis,S.oralis),血鏈球菌(Streptococcus sanguinis,S.sanguinis),格登鏈球菌(Streptococcus gordonii,S.gordonii)。本研究采用人體β防御素(human beta-defensins hBDs)抑菌,根據(jù)現(xiàn)有研究基礎(chǔ)及參考文獻(xiàn),為了最大限度的保留人體β防御素3(“human beta-defensins 3,hBD3”)的抑菌作用和生物穩(wěn)定性,本課題合成hBD3的3條短肽hBD3-1,hBD3-2,hBD3-3,以抑制生物膜初期附著菌生長(zhǎng),進(jìn)而抑制口腔生物膜的形成。本研究避免了抗菌肽刺激機(jī)體后產(chǎn)生的特異性免疫反應(yīng),防止了在高滲環(huán)境下抗菌肽失去抑菌活性,克服了藥物抗菌所誘導(dǎo)的耐藥性,為生成嵌合多肽,治療相關(guān)感染性疾病開(kāi)辟了新途徑。目的:hBD3為“天然抗生素”中的一員,在口腔中作為第一道防御屏障,能夠治療革蘭氏陽(yáng)性菌(Gram-positive,G+)和革蘭氏陰性菌(Gram-negative,G-)造成的感染性疾病。鏈球菌是牙菌斑形成過(guò)程中的初期附著菌,同時(shí)也是造成口腔牙菌斑疾病的重要原因。本論文設(shè)計(jì)合成hBD3的N端起始的三條短肽hBD3-1,hBD3-2,hBD3-3,評(píng)估hBD3-1,hBD3-2,hBD3-3對(duì)口腔鏈球菌的抑菌作用。方法:本研究中設(shè)計(jì)合成hBD3-1,hBD3-2,hBD3-3,檢測(cè)hBD3-1,hBD3-2,hBD3-3對(duì)S.oralis,S.sanguinis和S.gordonii生物活性的抑制作用。通過(guò)最小抑菌濃度(Minimal Inhibitory Concentration MIC),最小殺菌濃度(Minimal Bactericidal Concentration MBC)和生物膜形成實(shí)驗(yàn)測(cè)試hBD3-1,hBD3-2,hBD3-3對(duì)不同狀態(tài)下鏈球菌生長(zhǎng)的抑菌作用。拉曼光譜(Raman spectroscopy)和圓二色光譜(Circular Dichroism spectroscopy)研究hBD3-1,hBD3-2,hBD3-3二級(jí)結(jié)構(gòu)。通過(guò)激光共聚焦顯微鏡(Confocal Laser Scanning Microscopy CLSM)和電子掃描顯微鏡(Scanning Electron Microscopy SEM)觀察鏈球菌細(xì)胞形態(tài)。結(jié)果:hBD3-1,hBD3-2,hBD3-3對(duì)鏈球菌的生長(zhǎng)均具有抑制性,其中浮游狀態(tài)下的S.oralis和S.sanguinis生物膜對(duì)hBD3-3的藥物敏感性最強(qiáng)(P0.05)。Raman spectroscopy實(shí)驗(yàn)結(jié)果表明hBD3-1,hBD3-2,hBD3-3的二級(jí)結(jié)構(gòu)中均具有酰胺Ⅰ和β折疊。但是在hBD3-2,hBD3-3的二級(jí)結(jié)構(gòu)中未發(fā)現(xiàn)酪氨酸。CD spectroscopy實(shí)驗(yàn)結(jié)果表明在口腔環(huán)境中hBD3-1,hBD3-2,hBD3-3抑菌活性保持穩(wěn)定,不會(huì)因?yàn)闇囟鹊淖兓绊懸志饔?且不受二硫鍵的影響。通過(guò)CLSM鏡下觀察染色的鏈球菌生物膜發(fā)現(xiàn)作用于hBD3-3的細(xì)菌生物膜的凋亡細(xì)胞染色比例要多于對(duì)照組。SEM鏡下觀察到hBD3-3對(duì)鏈球菌生物膜生物量的減少和細(xì)菌形態(tài)的破壞。結(jié)論:hBD3-3作為hBD3 45氨基酸序列中的一段短肽,能夠有效抑制初期附著菌的生長(zhǎng),進(jìn)而抑制口腔生物膜的形成,最終預(yù)防和降低口腔生物膜感染性疾病,維持口腔健康。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R780.2

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本文編號(hào)：1791157