摘要
生物膜(biofilm)是由细菌和其分泌的胞外基质在物体表面形成的高度组织化的多细胞结构。一旦形成生物膜,细菌将具有极强的抗生素耐药性(比浮游细菌高100～1000倍)和逃避机体免疫系统攻击的能力。生物膜无处不在,在很多方面给人类生活带来严重的影响。根据美国NIH的调查公告,80%以上的细菌性感染与生物膜有关。由于传统的抗菌药物对细菌生物膜几乎没有作用,因此,如何防治细菌生物膜已成为人类面临的巨大挑战。
牙菌斑生物膜是牙周病的始动因素,在牙菌斑生物膜的刺激下,牙周组织细胞分泌的细胞因子在牙槽骨吸收及牙周软组织破坏的过程中发挥着重要作用。然而,目前绝大多数研究集中在浮游细菌对牙周组织细胞毒性的研究,而关于细菌生物膜与牙周病的研究极少。胞外多糖是细菌生物膜的主要成分,它决定了生物膜的结构和功能。最新研究表明,伴放线放线杆菌是侵袭性牙周炎的主要致病菌,它产生的β-1,6-N-乙酰葡聚糖胺是其生物膜的主要成份,在生物膜形成和致病性方面发挥着重要作用。因此,本论文旨在从海洋微生物中筛选降解β-1,6-N-乙酰葡聚糖胺的多糖水解酶,并研究其抗牙菌斑细菌生物膜的作用。
以β-1,6-N-乙酰葡聚糖胺为唯一碳源的选择性培养基,从黄海海泥样品中筛选到一株高产N-乙酰葡萄糖苷酶的菌株。经16S rRNA技术鉴定,该菌株被鉴定为假交替单胞菌属,命名为假交替单胞菌(Pseudoalteromonas sp.)QY403。
利用硫酸铵沉淀、离子交换层析和凝胶过滤层析技术,从菌株QY403的发酵液上清中分离纯化到一种N-乙酰葡萄糖苷酶NagY1。该酶的分子量为40.2 kD;酶反应的最适pH为6.0,pH稳定范围为5.0～7.2;最适反应温度为40℃,酶的活性在0～40℃之间基本稳定;Mg~(2+)、Ca~(2+)、Mn~(2+)、Ba~(2+)、Fe~(2+)、Fe~(3+)、EDTA对于酶的活性有促进作用,而Cu~(2+)、Ni~(2+)对于酶的活性有抑制作用,SDS可以使酶完全失去活性。酶的底物专一性分析结果表明该酶能特异性降解β-1,6-N-乙酰葡聚糖胺。
在上述研究的基础上,本文进一步研究了N-乙酰葡萄糖苷酶NagY1的抗A.a生物膜作用,并且从细胞因子产生的角度观察了NagY1对A.a致病性的影响。首先,利用A.a的体外生物膜模型,评价了不同浓度的NagY1对A.a生物膜形成的抑制作用和对已形成生物膜的清除作用。结果显示,NagY1对A.a生物膜形成具有显著的抑制作用(＞50%),并且对已形成生物膜的清除率为74.5%(P＜0.05)。其次,研究了A.a生物膜刺激对人牙周韧带细胞产生细胞因子RANKL、IL-6、IL-1β、IL-8、IFN-γ的影响以及N-乙酰氨基葡萄糖苷酶降解A.a生物膜对牙周韧带细胞产生细胞因子的影响。将A.a生物膜与原代培养的人牙周韧带细胞共培养0-9h,用实时荧光定量PCR技术检测人牙周韧带细胞的细胞因子mRNA的表达量,分别与浮游A.a刺激组以及空白对照组进行比较分析。实验结果显示,A.a生物膜和浮游菌刺激均可以提高人牙周韧带细胞的IL-1β、IL-8、IFN-γmRNA表达量;A.a生物膜比A.a浮游菌具有更强地刺激人牙周韧带细胞产生细胞因子RANKL、IL-6、IL-1β、IL-8、IFN-γmRNA的作用(P＜0.05)。N-乙酰葡萄糖苷酶NagY1降解A.a生物膜,使A.a生物膜刺激细胞人牙周韧带细胞产生细胞因子mRNA表达量较明显降低。
综上所述,本论文筛选得到一株高产N-乙酰氨基葡萄糖苷酶的海洋交替假单胞菌,并从其发酵上清液中成功地分离纯化了一种N-乙酰氨基葡萄糖苷酶NagY1;NagY1可以有效地抑制牙周病的常见致病菌A.a的生物膜形成和清除其已形成的生物膜;并且NagY1能显著地降低A.a生物膜引起的人牙周韧带细胞细胞因子mRNA表达量。本研究结果将为NagY1的临床应用提供理论基础,为牙周病等细菌生物膜相关疾病的防治开辟新途径。
Biofilm is a height organized multicellular structure which consisted with bacterium and its secretory extracellular matrix and formed on the surface of object. Once the biofilm formed,the bacteria will become more resistant to antibiotic(100～1000 times higher than planktonic cells) and to the aggressive attack of immune system.The common existence of biofilm gives great trouble to the human beings life in many ways.According to the public announcement of NIH of America,more than 80%of bacterial infection concerned with biofilm.Because of the nearly ineffective of traditional antibacterials to the bacterial biofilm,thus,how to get rid of biofilm becomes a great challenge of human beings.
Biofilm of dental plaque is the initiating agent of periodontal disease.At the stimulus of biofilm of dental plaque,the cytokine of periodontal tissue secretory plays an important role in the process of frontal resorption and periodontal histoclasia. However,the present majority study only focused on the cytotoxic of periodontal tissue induced by bacteriophankton.There's very few study at the role of bacterial biofilm to participate in periodontal disease.Extracellular polysaccharide is the major component of bacterial biofilm and determines the structure and function of biofilm. Actinobacillus actinomycetemcomitans(A.α) is the main pathogenic bacterium of invasive periodontitis.β-1,6-N-acetylglucosamine produced by A.αplays an important role in the structure and function of the biofilm.Therefore,this study wants to findβ-1,6-N-acetylglucosaminidase from ocean microorganisms and to study it's effects on A.αbiofilm.
A marine bacterium strain QY403,which has a high yield ofβ-1,6-N- acetylglucosaminidase,is isolated from yellow sea mud sample using medium containingβ-1,6-N-acetylglucosamine as the sole carbon source.Strain QY403 is identified to belong to genus Pseudoalteromonas using molecular phylogenetic analysis based on the partial 16S rRNA gene sequence.
Aβ-1,6-N-acetylglucosaminidase is purified from culture supernatants of strain QY403 after ammonium sulfate precipitation,ion exchange chromatography and gel filtration chromatography procedure.Molecular weight of this enzyme,which is named by NagY1,is 40.2 kDa.NagY1 is most active at 40℃and at pH6.0 and is stable below 40℃and over a range of pH5.0-7.2.Mg~(2+),Ca~(2+),Mn~(2+),Ba~(2+),Fe~(2+), Fe~(3+) and EDTA have the promotive effect on the enzymatic activity,while Cu~(2+) and Ni~(2+) have depressant effect,SDS has the deactivation effect.Substrate specificity analytic result of this enzyme showes that it only degradsβ-1,6-N-acetylglucosamine.
On the foundation of the previous research,the effects of NagY1 on Actinobacillus actinomycetemcomitans(A.α) biofilm and cytokine production are investigated.At first,using the extra biofilm model of A.αto appraise the inhibition effect of different concentration of NagY1 on A.αbiofilm formation and detachment effect on the existed A.αbiofilm.The results show a prominent inhibition effect of NagY1 on A.αbiofilm formation(＞50%) and a detachment rate to the existed A.αbiofilm of 74.5%(P＜0.05).Then the effects of A.αbiofilm and A.αbiofilm degraded by NagY1 on the cytogenic of RANKL,IL-6,IL-1β,IL-8,IFN-γfrom the human periodontal ligament cells are investigated.The results show that both of biofilm cells and planktonic cells of A.αcan enhance the expression of IL-1β,IL-8 and IFN-γin human periodontal ligmament cells.The A.αbiofilm has more strong stimulation effect on cytogenic of RANKL,IL-6,IL-1β,IL-8 and IFN-γthan planktonic cells (P＜0.05).NagY1 can degrade A.αbiofilm and decrease comparatively cytokine mRNA expression.
In summary,this study isolates one marine strain Pseudoalteromonas sp.QY403, and purified aβ-1,6-N-acetylglucosaminidase NagY1 from culture supernatants. NagY1 can effectively restrain A.αbiofilm formation and detach the existed biofilm of periodontal disease.Further more,NagY1 can remarkably decrease the cytogenic mRNA expression of human periodontal ligament cell.The results of this study can give rationale to the clinical use of NagY1,and open up a new approach to the prevention and cure of bacterium biofilm related periodontal disease.
引文
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