【作者】 宋玮；

【导师】 肖明振； 田宇；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2008， 博士

【摘要】 牙髓、根尖周炎症是口腔内科常见病,细菌则是牙髓、根尖周病的主要致病因素。根管治疗术是通过根管预备、根管消毒和根管充填等步骤,杀灭感染细菌,消除来自根管的刺激源,利用根尖周组织血运丰富、修复再生能力强的特点,治疗牙髓坏死或根尖周病的方法。其目标是从根管开口到缩窄的根尖孔对整个根管进行三维清洁、成形和封闭,以达到治愈患牙,保存并恢复其功能的作用。而根管系统本身结构复杂且与根尖周、牙周相通,虽然经过机械和化学预备后,根管内的细菌、坏死牙髓和根管内壁的感染物,大部分已被清除,但是对于侧壁牙本质小管深部,侧支根管和根尖周等,器械以及冲洗液不易到达的地方仍存在细菌、毒素等病原刺激物,所以根管清理后的灭菌消毒仍是不容忽视的。长期的临床追踪观察和基础研究形成了很多不同的根管消毒方法和消毒药物,目前学者们正在试图寻找一种较理想的根管消毒药物。β防御素-3作为一种内源性抗菌肽,其快速、高效、广谱的抗菌效能和较少产生耐药性的特点,使得其在口腔医学中的研究和应用有着较大的发展空间和良好的发展前景,将其引入感染根管的治疗领域,对根管内的致病菌产生杀菌作用,将有可能大大提高和改善根管内灭菌消毒的效果,达到治愈牙髓、根尖周病变的目的。本课题本着基础研究与临床应用并重的目的而展开,通过基因工程技术来获得有活性的重组人防御素-3,并分析其抗菌能力,经过感染根管的体外和动物体内实验进一步研究rhBD-3在感染根管治疗中的作用,以期为牙髓和根尖周感染,尤其是根管内的重度感染的治疗寻找一条新的途径。所进行的研究及结果如下:第一部分hBD3基因的克隆和序列测定本研究根据在GenBank中登录的hBD3的完整的cDNA序列,设计一对引物,以临床获取的正常人新鲜牙龈组织的mRNA为模板,采用RT-PCR的方法获得了hBD3成熟肽编码区全长序列,将其克隆入pGEM-T-EASY载体,在大肠杆菌中进行扩增,并进行了序列测定。结果表明,我们所获得的序列与Harder等文献报道的序列完全一致,未发现有碱基的缺失或突变。第二部分hBD3在大肠杆菌中的表达、纯化及其表达产物的活性检测将hBD3成熟肽基因成功地正向克隆入pET-32a载体,构建融合表达载体。将含有pET-32a-hBD3重组质粒的BL21菌株经IPTG诱导后,表达产物经SDS-PAGE显示在约24KD处看到预期的条带。Western blot结果也提示HIS融合蛋白有基础性表达。对不同诱导时间、温度、IPTG浓度时融合蛋白表达率分析的结果表明:hBD3在35℃、0.6mmol/L IPTG、诱导6小时表达量分别达高峰。表达的融合蛋白大部分以包涵体形式存在于沉淀中,小部分以可溶性形式存在于上清中。包涵体溶解物过Ni-NTA柱亲和层析纯化,经洗脱液洗脱收集得到目的蛋白,然后经多步透析法复性后,再进行离子交换层析和凝胶柱脱盐、浓缩。以SDS-PAGE检测,纯化蛋白为单一条带。肠激酶可以对融合蛋白进行切割,rhBD3从融合蛋白形式中分离出来,大小约5.1KD,与理论值接近。rhBD3的生物学活性分析实验显示rhBD3融合蛋白和融合蛋白酶切后的rhBD3成熟肽对于金黄色葡萄球菌和大肠杆菌都有明显抑菌环出现,表示均有抗菌活性。第三部分重组hBD3对染根管致病菌抗菌活性的体外研究重组hBD3对感染根管各种致病菌的抗菌活性检测结果显示,rhBD3对所有试验菌株均有抑制和杀灭作用,随着rhBD3的浓度增加,细菌数量减少,与天然提取的hBD3活性基本一致。但是MIC或MBC浓度对不同的细菌而言并不一致。rhBD3除了对常见兼性厌氧的金黄色葡萄球菌,大肠杆菌有作用以外,对根管内常见的专性厌氧菌的三种标准株有较好的抑制作用,而且对内氏放线菌、白色念珠菌、粪肠球菌也有作用,对变形链球菌的抑制作用则比较小。从重组hBD3对离体人牙根管内致病菌抗菌活性研究的结果分析可以看出,封药后细菌阳性的根管数量明显低于封药前,其差异均有统计学意义(P<0.005),说明四种消毒药物包括rhBD3对根管内的优势致病菌均有抗菌或抑菌作用;对比四种消毒药物对各种细菌的抗菌活性发现,其中FC组、碘仿糊剂组和rhBD3组的抗菌活性高于Ca(OH)2糊剂组(P<0.05),特别是对于厌氧菌;但是FC组、碘仿糊剂组和rhBD3组间的抗菌活性则无明显区别(P>0.05)。第四部分重组hBD3对犬的感染根管内致病菌抗菌活性的体内研究本实验建立了犬的感染根管动物模型,摄X线片证实各实验犬牙出现不同程度的牙周膜腔增宽、根尖周骨质稀疏区。实验中各处理组在根管封药后,无论厌氧还是需氧菌,细菌的检出率明显减少,经x2检验,消毒前、后差异有统计学意义(P<0.005)。进一步两两比较,Fc组,Ca(OH)2糊剂组,rhBD3组均与空白对照组有显著差别(P<0.05),前三者的细菌检出数值远小于后者,说明Fc,Ca(OH)2糊剂,rhBD3对根管内的需氧和厌氧菌均有抑制作用,都能显著减少预备后根管内的细菌。以上结果证实了rhBD3在感染根管内的抑菌效果。另外Fc组、rhBD3组与Ca(OH)2糊剂组也有显著性差别(P<0.05),从数值上看,前两者的效果好于后者。经x2检验,,rhBD3组对需氧和厌氧菌消毒前后的差异均具有极显著性(P<0.005),说明消毒效果都非常好,而对需氧和厌氧菌2个菌种之间的消毒差异无显著性(P>0.05)。切片观察封药后各组实验牙根尖周组织炎症分级情况,空白对照组与3个药物处理组有统计学差别(P<0.05),说明每个药物处理组对根尖周炎症均有一定影响。Fc组与Ca(OH)2糊剂组相比有显著性差别(P <0.05),从结果来看,Fc组的根尖周组织炎症程度重于Ca(OH)2糊剂组,提示Fc组对根尖周炎症的作用不理想,这可能与其本身的强刺激性有关。而rhBD3组与Ca(OH)2糊剂组相比也有显著性差别,这或许与β-防御素具有间接的免疫激活作用有关。综上所述,根管系统的复杂性和感染细菌的多样性,决定了根管灭菌消毒的必要性。根管预备完成后,在根管内封入适当的药物,可以进一步控制微生物,防止再感染,从而巩固和加强根管预备的效果。根据以往对β防御素的研究结果结合以上我们的实验,提示rhBD3用于感染根管内的杀菌消毒,具有一定的临床可行性,有很好的应用前景,其具体的多方面的应用还需要进一步的实验研究。更多还原

【Abstract】 Pulpitis and periapical periodontitis are two common diseases in dental clinic. Microorganisms are major etiological agents in pulpal and periapical disease. Root canal therapy is a method of treating pulp necrosis and periapical diseases with the help of periapical tissues which are characterized by abundant blood flow and strong ability of repair and regeneration. The main goal of endodontic therapy is the elimination of microorganism from the root canal system and the prevention of subsequent reinfection, so as to cure the inflammation and conserve the sick tooth. But the persistence or reinfection of bacteria in the complicated root canal system often leads to failure of root canal treatment. Although most of the bacteria, necrotized pulp and infected dentin are removed from the root canals after chemical and mechanical preparation, there still have etiological agents such as residual bacteria, endotoxin in collateral pulp canals, deep part of dentinal tubules and periapex. Therefore, root canal disinfection is not a single procedure can be omitted. There are many different endodontic sterilizing medicines after longtime clinic and research work. Therefore, to thoroughly kill and avoid again infection of these microorganisms, endodontic sterilizing medicine should ideally have a powerful and broad-spectrum antibacterial capability.Humanβ-defensin-3 (hBD3) are a novel human epithelial-derived endogenous cationic antimicrobial peptides, which exhibits a strong, quickly and broad spectrum bactericidal action, in addition, with low susceptibility to acquired bacterial resistance. Consequently, the antibacterial properties of hBD3 have attracted the attention of researchers in the field of dentistry. However, there is nothing in the literature regarding the antibacterial efficacy of hBD3 against bacteria in the root canals. The aim of this study was to obtain recombinant hBD3 by means of gene engineering technique, moreover, evaluate and compare the antimicrobial effectiveness of hBD3 with other regular intracanal medicaments, so as to develop a new antimicrobial agent that suitable to treat pulp and periodontal inflammation, especially, severe infections. Our research and outcomes:1. Cloning of hBD3 cDNA from human gingival epithelium Two primers were designed based on hBD3 nucleotide sequence data registered in GenBank and used to clone hBD3-specific sequence from the mRNA template, which obtained from normal human gingival epithelium tissue’s total RNA. After the RT-PCR, the product of expected size was inserted into cloning vector pGEM-T. Recombinant pGEM-T / HBD-3 vector was transduced into competent cell DH5αfor amplification, followed by sequencing. The result display that hBD3 gene was successfully cloned from human gingival tissue. Agarose gel electrophoresis showed an expected single strap about 250 bp and sequence of the product was coincident with the cDNA of hBD3 nucleotide order (GenBank TM accession nos.AJ237673).2. Expression of hBD3 in E. coli, purification and antimicrobial bioactivity assayhBD-3 gene was ligated to vector pET-32a(+). The expression vector was constructed, called pET32a/ HBD-3, which was finally transformed into E. coli BL21 (DE3) following the standard procedure. The foreign protein expression was induced by adding IPTG to transformed monoclone. According to SDS-PAGE analysis, the target protein was successfully expressed. A molecular weight of about 24 kd fusion protein was obtained, and was further confirmed by western blotting analysis. Expression optimization: the maximum was obtained when the expression condition is 35℃、0.6mmol/L IPTG and 6 hours induction.Cytorrhyctes, products of the massive expression, were dissolved by guanidine hydrochloride. After metal-affinity chromatography, rhBD3, with the 6Histagged fusion protein, was renatured by means of dilution for protein refolding. Then, rhBD3 was further purified by cationic exchange chromatography and desalted by gel chromatography. The SDS-PAGE assay showed the results. The mature peptide was obtained from fusion protein after cut by enterokinase, and about 5.1kd, similar with the reference.The antimicrobial bioactivity assay of recombinant rhBD3 display that both the fusion protein and the mature peptide exhibit antimicrobial activity against S. aureus and E. coli.3. In vitro study of the antibacterial activity of recombinant hBD3 against pathogenic bacterium from the infected root canalsSome distinct pathogenic bacterium (including anaerobe and facultative anaerobe) in endodontic infections: F. nucleatum, B. melanogenicus, P. anaerobius, S. mutans, A. naeslundii, E. faecalis, C. albicans, S. aureus and E. coli were performed in the antimicrobial activity assay. Assess the ability of peptides to kill bacteria, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were obtained using a conventional microdilution method. Results presented that the recombination protein exhibits a broad spectrum antimicrobial activity. As the colony of bacteria remaining illustrated that the growth of bacteria was dramatically suppressed by rhBD3. And MIC and MBC of each bacterium were different respectively. Analyses of the in vitro antimicrobial properties of rhBD3 revealed antimicrobial activity against common facultative anaerobes: S. aureus and E. coli as well as three potential pathogenic anaerobes in endodontic infections: F. nucleatum, B. melanogenicus, and P. anaerobius. Furthermore, rhBD3 kills A. naeslundii, C. albicans and vancomycin-resistant E. faecium at similar low concentrations. But it has not so effective antimicrobial activity against S. mutans.Next, the study of the antibacterial activities of rhBD3 on pathogenic bacterium from the infected root canals in vitro showed that all experimental medicaments were effective against microorganism after sealed in the infected root canals. A statistically significant difference was found between the experimental groups and negative control groups (P<0.005). Among all of the medicament groups, FC Group, iodoform Group and rhBD3 Group were found to be more effective than Ca(OH)2 Group against anaerobes (P<0.05), however, no significant difference was found between FC Group, iodoform Group and rhBD3 Group (P>0.05). Significantly, the final product——rhBD3 can markedly kill and inhibit the multiplication of bacteria, especially anaerobe, in root canals.4. In vivo study of rhBD3 used as an intracanal medicament on infected dog’s root canalsThe animal model of infected root canal was made to evaluate the antibacterial activities on pathogenic strains in the root canals. X-ray picture confirm the existence of periapical periodontitis in dog’s incisors. The results showed that comparing with control group, the detection rate of bacteria, no matter anaerobes or aerobes, in experiment groups (Fc, Ca(OH)2 paste, rhBD3) decrease obviously. X2 analysis showed a statistically significant difference between the experimental groups and negative control groups (P<0.05). They all can reduce the amount of strains in root canals after sealing. Among all of the medicament groups, FC Group and rhBD3 Group were found to be more effective than Ca(OH)2 Group, especially against anaerobes (P<0.05).The HE slice was also made to evaluate the influence on the inflammation of periapical disease among four different groups. All the medicaments have influence on periapical inflammation. A statistically significant difference was found between the experimental groups and negative control groups (P<0.05).The level of inflammation of FC Group was found to be more serious than Ca(OH)2 Group (P<0.05). We conclude that it may due to the intense cytotoxicity. However, significant difference was found between rhBD3 Group and Ca(OH)2 Group (P<0.05). So, rhBD3 can also alleviate the inflammation of periapical disease to some extent, but not so good as Ca(OH)2 paste, which may be related to its indirect immune activation.In conclusion, hBD3, obtained by gene engineering technique, exhibits a broad spectrum antibacterial activity, at low micromolar concentrations, especially to many critical infected pathogenic microbes in root canals. The results indicate that rhBD3 has the potential to eliminate bacteria in the local microenvironment of the root canal system, thus lead to the success of endodontic treatment. The prospect of the application of rhBD3 as an intracanal medicament is good, and more deep research should be done in the future.更多还原