人β防御素3的重组表达和在感染根管治疗中抑菌作用的初步实验研究
|
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。
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 assay
hBD-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 canals
Some 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 canals
The 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.
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。
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 assay
hBD-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 canals
Some 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 canals
The 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.
引文
[1] Chertov O, Yang D and Oppenheim. Leukocyte granule proteins mobilize innate host defenses and adaptive immune responses. Immunological Reviews. 2000; 177: 68-78.
[2] 李建民,周开亚,张双全。先天免疫中的抗菌肽及其进化。南京师大学报(自然科学版),1999;22:16.
[3] Boman HG. Antibacterial peptides: key components needed in immunity. Cell. 1991;65:205-207.
[4] 张继南,陈红霞。抗菌肽及其应用研究进展。 生物技术通讯,2006;17:669-672.
[5] Lehrer RI, Ganz T, Selsted ME. Defensins: endogenous antibiotic peptides of animal cells. Cell. 1991;64:229-230.
[6] Kimbrell DA. Insect antibacterial proteins: not just for insects and against bacteria. Bioessays. 1991;13:657-663.
[7] Hoffmann JA, Hetru C. Insect defensins: inducible antibacterial peptides. Immunol Today. 1992;13:411-415.
[8] Kagan BL, Ganz T, Lehrer RI. Defensins: a family of antimicrobial and cytotoxic peptides. Toxicology. 1994;87(1-3):131-149.
[9] Hultmark D. Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet. 1993;9:178-183.
[10] Bensch KW, Raida M, M?gert HJ, Schulz-Knappe P, Forssmann WG. hBD-1: a novel beta-defensin from human plasma. FEBS Lett. 1995;368:331-335.
[11] 李春丽,郑振宇,赵卫东。人防御素的研究进展及其应用前景。中国生物制品学杂志,2007;20:146-148.
[12] Schroder JM. Epithelial antimicrobial peptides: innate local host response elements. Cell Mol Life Sci. 1999;56:32-46.
[13] Garcia-Olmedo F, Rodriguez-Palenzuela P, Molina A, Alamillo JM, López-Solanilla E, Berrocal-Lobo M, Poza-Carrión C. Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence. FEBS Lett. 2001;498:219-222.
[14] Broekaert WF, Terras FR, Cammue BP, Osborn RW. Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol. 1995;108:1353-1358.
[15] Broekaert WF, Cammue BPA, De Bolle MFC, et al. Antimicrobial peptides from plants. Crit Rev Plant Sci. 1997;16:297-323.
[16] Terras FR, Torrekens S, Van Leuven F, Osborn RW, Vanderleyden J, Cammue BP, Broekaert WF. A new family of basic cystein-rich antifungal proteins from Brassicaceae species. FEBS Lett. 1993;16:233-240.
[17] Steiner H, Hultmark D, Engstr?m A, Bennich H, Boman HG. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature. 1981;292:246-248.
[18] Fehlbaum P, Bulet P, Michaut L, Lagueux M, Broekaert WF, Hetru C, Hoffmann JA. Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. J Biol Chem. 1994;269:33159-33163.
[19] Lee JY, Edlund T, Ny T, Faye I, Boman HG. Insect immunity. Isolation of cDNA clones corresponding to attacins and immune protein P4 from Hyalophora cecropia. EMBO J. 1983;2:577-581.
[20] Lamberty M, Zachary D, Lanot R, Bordereau C, Robert A, Hoffmann JA, Bulet P. Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect. J Biol Chem. 2001;276:4085-4092.
[21] Imler JL, Tauszig S, Jouanguy E, Forestier C, Hoffmann JA. LPS-induced immune response in Drosophila. J Endotoxin Res. 2000;6:459-462.
[22] Tang YQ, Yuan J, Osapay G, Osapay K, Tran D, Miller CJ, Ouellette AJ, Selsted ME. A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science. 1999;286:498-502.
[23] Tran D, Tran PA, Tang YQ, Yuan J, Cole T, Selsted ME. Homodimeric theta-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides. J Biol Chem. 2002;277:3079-3084.
[24] Bals R. Epithelial antimicrobial peptides in host defense against infection. Respir Res. 2000;1:141-150.
[25] Boman HG. Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scand J Immunol .1998;48:15-25.
[26] Panyutich AV, Panyutich EA, Krapivin VA, Baturevich EA, Ganz T. Plasma defensin concentrations are elevated in patiens with septicemia and baterial meningitis. J Lab Clin Med. 1993;122:202-207.
[27] van Wetering S, Sterk PJ, Rabe KF, Hiemstra PS. Defensins: key players or bystanders in infection, injury, and repair in the lung. J Allergy Clin Immunol. 1999;104:1131-1138.
[28] Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T, Tack BF, McCray PB Jr. Productions of beta-defensins by human airway epithelia. Proc Natl Acad Sci USA.1998;95:14961-14966.
[29] Lillard JW Jr, Boyaka PN, Chertov O, Oppenheim JJ, McGhee JR. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc Natl Acad Sci USA. 1999;96:651-656.
[30] van Wetering S, Mannesse-Lazeroms SP, Dijkman JH, Hiemstra PS. Effect of neutrophil serine proteinases and defensins on lung epithelial cells:modulation of cytoxicity and IL-8 production. J Leukoc Biol. 1997;62:217-226.
[31] Diamond G, Zasloff M, Eck H, Brasseur M, Maloy WL, Bevins CL. Tracheal antimicrobial peptide, a novel cystein-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA. Proc Natl Acad Sci USA. 1991;88:3952-3956.
[32] Harder J, Bartels J, Christophers E, Schr?der JM. A peptide antibiotic from human skin. Nature. 1997;387:861.
[33] Schutte BC, Mitros JP, Bartlett JA, Walters JD, Jia HP, Welsh MJ, Casavant TL, McCray PB Jr. Discovery of five conserved beta-defensin gene clusters using a computational search strategy. Proc Natl Acad Sci USA. 2002;99:2129-2133.
[34] Harder J, Siebert R, Zhang Y, Matthiesen P, Christophers E, Schlegelberger B, Schr?der JM. Mapping of the gene encoding human beta-defensin-2 (DEFB2) to chromosome region 8p22-p23.1. Genomics. 1997;46:472-475.
[35] Liu L, Wang L, Jia HP, Zhao C, Heng HH, Schutte BC, McCray PB Jr, Ganz T. Structure and mapping of the human beta-defensin HBD-2 gene and its expression at sites of inflammation. Gene. 1998;222:237-244.
[36] Jia HP, Schutte BC, Schdy A, Linzmeier R, Guthmiller JM, Johnson GK, Tack BF, Mitros JP, Rosenthal A, Ganz T, McCray PB Jr. Discovery of newhuman beta-defensins using a genomics-based approach. Gene. 2001;263:211-218.
[37] Harder J, Bartels J, Christophers E, Schroder JM. Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic. J Biol Chem. 2001;276:5707-5713.
[38] Garcia JR, Krause A, Schulz S, Rodríguez-Jiménez FJ, Klüver E, Adermann K, Forssmann U, Frimpong-Boateng A, Bals R, Forssmann WG. Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of anti-microbial activity. FASEB J. 2001;15:1819-l821.
[39] Oppenheim JJ, Biragyn A, Kwak LW, Yang D. Roles of antimierobial peptides such as defensins in innate and adaptive immunity. Ann Rheum Dis. 2003;62:1117-1121.
[40] Dhople V, Krukemeyer A, Ramamoorthy A. The human beta-defensin-3, an antibacterial peptide with multiple biological functions. Biochimica et Biophysica Acta. 2006;1758:1499-1512.
[41] Valore EV, Park CH, Quayle AJ, Wiles KR, McCray PB Jr, Ganz T. Human beta-defensin-1: an antimicrobial peptide of urogenital tissues. J Clin Invest. 1998;101:1633-1642.
[42] Bals R, Wang X, Wu Z, Freeman T, Bafna V, Zasloff M, Wilson JM. Human beta-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung. J Clin Invest. 1998;102:874-880.
[43] Diamond G, Jones DE, Bevins CL. Airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene. Proc Natl Acad Sci USA. 1993;90:4596-4600.
[44] Ganz T. Defensins and host defense. Science. 1999;286:420-421.
[45] Hoover DM, Rajashankar KR, Blumenthal R, Puri A, Oppenheim JJ, Chertov O, Lubkowski J. The structure of human beta-defensin-2 shows evidence of higher order oligomerization. J Biol Chem. 2000;275:32911-32918.
[46] Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schr?der JM, Wang JM, Howard OM, Oppenheim JJ. Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science. 1999;286:525-528.
[47] Solomon S, Hu J, Zhu Q, Belcourt D, Bennett HP, Bateman A, Antakly T. Corticostatic peptides. J Steroid Biochem Mol Biol. 1991;40:391-398.
[48] Becker MN, Diamond G, Verghese MW, Randell SH. CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem. 2000;275:29731-29736.
[49] Hao HN, Zhao J, Lotoczky G, Grever WE, Lyman WD. Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines. J Neurochem. 2001;77:1027-1035.
[50] Abiko Y, Suraweera AK, Nishimura M, Arakawa T, Takuma T, Mizoguchi I, Kaku T. Differential expression of human beta-defensin-2 in keratinized and non-keratinized oral epithelial lesions; imunohis tochemistry and in situ hybridization. Virchows Arch. 2001;438:248-253.
[51] Mizukawa N, Sawaki K, Yamachika E, Fukunaga J, Ueno T, Takagi S, Sugahara T. Presence of human beta-defensin-2 in oral squamous cell carcinoma. Anticancer Res. 2000;20:2005-2007.
[52] Wada A, Mori N, Oishi K, Hojo H, Nakahara Y, Hamanaka Y, Nagashima M, Sekine I, Ogushi K, Niidome T, Nagatake T, Moss J, Hirayama T. Induction of human beta-defensin-2 mRNA expressi on by Helicobacterpylori in human gastric cell line MKN45 cells on cag pathogeni city island. Biochem Biophys Res Commun. 1999;263:770-774.
[53] Abiko Y, Mitamura J, Nishimura M, Muramatsu T, Inoue T, Shimono M, Kaku T. Pattern of expression of beta-defensins in oral squamous cell carcinoma. Cancer Lett. 1999;143:37-43.
[54] Tomita T, Hitomi S, Nagase T, Matsui H, Matsuse T, Kimura S, Ouchi Y. Effect of ions on antibacterial activity of human beta-defensin-2. Microbiol Immunol. 2000;44:749-754.
[55] Duits LA, Rademaker M, Ravensbergen B, van Sterkenburg MA, van Strijen E, Hiemstra PS, Nibbering PH. Inhibition of hBD-3, but not hBD-1 and hBD-2, mRNA expression by corticosteroids. Biochem Biophys Res Commun. 2001;280:522-525.
[56] Ganz T. Defensins and other antimicrobial peptides: a historical perspective and an update. Comb Chem High Throughput Screen. 2005;8:209-217.
[57] Batoni G, Maisetta G, Esin S, Campa M. Human beta-defensin-3: a promising antimicrobial peptide. Mini-Reviews in Medicinal Chemistry. 2006;6:1063-1073.
[58] Schmid P, Grenet O, Medina J, Chibout SD, Osborne C, Cox DA. An intrinsic antibiotic mechanism in wounds and tissue-engineered skin. J Invest Dermatol. 2001;116:471-472.
[59] Kisich KO, Heifets L, Higgins M, Diamond G. Antimycobacterial agent based on mRNA encoding human beta-defensin-2 enables primary macrophages to restrict growth of mycobacterium tuberculosis. Infect Immun. 2001;69:2692~2699.
[60] Schibli DJ, Hunter HN, Aseyev V, Starner TD, Wiencek JM, McCray PB Jr, Tack BF, Vogel HJ. The solution structures of the human β-defensins lead toa better understanding of the potent bactericidal activity of HBD3 against Saureus. J Biol Chem, 2002;277:8279-8289.
[61] Joly S, Organ C, Johnson GK, McCray PB Jr, Guthmiller JM. Correlation between β-defensin expression and induction profiles in gingival keratinocytes. Mol Immunol. 2005;42:1073-1084.
[62] Harder J, Meyer-Hoffert U, Wehkamp K, et a1. Differential gene induction of human β-defensins (hBD1,-2,-3,and-4) in keratinocytes is inhibited by retinoic acid. J Invest Dennatol. 2004;123:522-529.
[63] Kragol G, Lovas S, Varadi G, Condie BA, Hoffmann R, Otvos L Jr. The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry. 2001;40:3016-3026.
[64] Hancock RE, Diamond G. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 2000;8:402-410.
[65] Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Imnmnol. 2003;3:710-720.
[66] Quinones-Mateu ME, Lederman MM, Feng Z, Chakraborty B, Weber J, Rangel HR, Marotta ML, Mirza M, Jiang B, Kiser P, Medvik K, Sieg SF, Weinberg A. Human epithelial beta-defensins 2 and 3 inhibit HIV-l replication. AIDS. 2003;17:F39-48.
[67] Wang Z, Wang G. APD: the Antimicrobial Peptide Database. Nucleic Acids Res. 2004;32(Database issue):D590-592.
[68] 司利钢,刘玺诚,李文梅,吕有勇,饶小春,马渝燕。大蒜素对肺癌A549细胞株β防御素表达的影响。首都医科大学学报,2006;27:792-794.
[69] Dunsche A, Acil Y, Siebert J, Harder J, Schr?der JM, Jepsen S. Expression profile of human defensins and antimicrobial proteins in oraltissues. J Oral Pathol Med. 2001;30:154-158.
[70] 钟德钰,杨美薷,吴琦,王伯瑶。人中性粒细胞防御素对龈下微生物的影响。华西口腔医学杂志,1997;15:123-125.
[71] Miyasaki KT, Lehrer RI. Beta-sheet antibiotic peptides as potential dental therapeutics. Int J Antimicrob Agents. 1998;9:269-280.
[72] Miyasaki KT, Iofel R, Huynh T, Huynh T, Lehrer RI. Killing of Fusobacterium nucleatum, Prophyromona gingivalis and Prevotella intermedia by protegrins. J Periodont Res. 1998;33:91-98.
[73] 刘进,奚 涛。人β防御素-3的研究进展。药物生物技术. 2005;12:337-341.
[74] 彭力,徐志南,方向明,吴金民; 岑沛霖。人防御索的研究进展。生物工程学报. 2003;19:261-266.
[75] Sahly H, Schubert S, Harder J, Rautenberg P, Ullmann U, Schr?der J, Podschun R. Burkholderia is highly resistant to human Beta-defensin-3. Antimicrob Agents Chemother. 2003;47:1739-1741.
[76] 扈英伟,朱敏,刘正。乳牙感染根管的菌丛分析。上海口腔医学,1998;7:143-146.
[77] Sato T, Hoshino E, Uematsu H, et al. Predominant obligate anaerobes in necrotic pulps of human deciduous teeth. Microbial Ecology in Health and Disease. 1993;6:269-275.
[78] Lu Q, Samaranayake LP, Darveau RP, Jin L. Expression of human beta-defensin-3 in gingival epithelia. J Periodontal Res. 2005;40:474-481.
[79] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning, a laboratory manual. 3nd ed. NewYork: Cold Spring Harbor Laboratory Press, 2002.
[80] 汪 家 政 , 范 明 。 蛋 白 质 技 术 手 册 , 北 京 : 科 学 出 版 社 ,2000;8:82-92;169-178.
[81] Schagger H, Von Jagow G. Tricine-sodium dodecyl sulfatepolyacrylamidegel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166:368-379.
[82] 肖晓蓉。口腔微生物学及实用技术,北京:北京医科大学出版社,1993;12:178;165-167.
[83] Goeddel DV. Systems for heterologous gene expression. Methods Enzymol, 1990;185:3-7.
[84] Balbas P, Bolivar F. Design and construction of expression plasmid vectors in Escherichia coli. Methods Enzymol, 1990;185:14-37.
[85] Hockney RC. Recent developments in heterologous protein production in Escherichia coli. Trends-Biotechnol, 1994;12:456-463.
[86] 卢圣栋。现代分子生物学实验技术。第二版,北京:中国协和医科大学出版社。1999:365-387.
[87] Hughes KT, Maloy SR. Use of operon and gene fusions to study gene regulation in Salmonella. Methods Enzymol. 2007;421:140-158.
[88] Hoover DM, Wu ZB, Tucker K, Lu W, Lubkowski J. Antimierobial characterization of humanβ-denfensin 3 derivatives. Antimlerob Agents Chemother, 2003;47:2804-2809.
[89] Forstner M, Leder L, Mayr LM. Optimization of protein expression systems for modern drug discovery. Expert Rev Proteomics. 2007;4:67-78.
[90] 随广超,胡美浩。影响大肠杆菌外源基因表达的因素。生物化学与生物物理进展。1994;21:128-132.
[91] 陈姗,何风田,董燕麟,李蓉芬,高会广,陈敏,彭家和。人β防御素3融合蛋白在大肠杆菌中的表达、纯化和活性分析。生物工程学报,2004;20:490-495.
[92] Rosa OP, Torres SA, Ferreira CM, Ferreira FB. In vitro effect of intracanal medicaments on strict anaerobes by means of the broth dilution method.Pesqui Odontol Bras. 2002;16:31-36.
[93] 肖晓蓉。口腔微生物学及实用技术,北京:北京医科大学出版社,1993;12:178;160-165.
[94] Gomes BP, Drucker DB, Lilley JD. Associations of specific bacteria with some endodontic signs and symptoms. Int Endod J. 1994;27:291-298.
[95] Gomes BP, Pinheiro ET, Gadê-Neto CR, Sousa EL, Ferraz CC, Zaia AA, Teixeira FB, Souza-Filho FJ. Microbiological examination of infected dental root canals. Oral Microbiol Immunol. 2004;19:71-76.
[96] Yoshida M, Fukushima H, Yamamoto K, Ogawa K, Toda T, Sagawa H. Correlation between clinical symptoms and microorganisms isolated from root canals of teeth with periapical pathosis. J Endod. 1987;13:24-28.
[97] 邓惠妹,赵雪梅。急性根尖周炎患牙根管内厌氧菌的细菌学研究。华西口腔医学杂志,1993;11:148-150.
[98] 梁景平,刘正,张国驰。感染根管厌氧菌的初步研究。中华口腔医学杂志,1991,26:28-30.
[99] Siqueria JF, Goncalves RB. Antibacterial activities of root canal sealers against selected anaerobic bacteria. Journal of Endod. 1996;22:89-90.
[100] Nakajo K, Nakazawa F, Iwaku M, Hoshino E. Alkali-resistant bacteria in root canal systems. Oral Microbiol Immunol. 2004;19:390-394.
[101] 章魁华,于世凤。实验口腔病理学。北京:人民卫生出版社,2002.29-31.
[102] 肖晓蓉。口腔微生物学及实用技术,北京:北京医科大学出版社,1993;12:95-98.
[103] 吉男匡宏 . 新しぃ概念に基づく根管治疗の实际 . 齿界展望 . 2001;97:805-810.
[104] 王伯沄,李玉松,黄高焺,张远强。病理学技术。北京:人民卫生出版社,2000.73
[105] Barbosa SV, Araki K, Spangberg LS. Cytotoxicity of some modified root canal sealers and their leachable components. Oral Surg Oral Med Oral Pathol, 1993;75:357-361.
[106] 张真,卢晓风。生物材料有效性和安全性评价的现状和趋势。生物医学工程杂志,2002;l9:ll7.
[107] Tziafas D, Alvanou A, Papadimitriou S. Effects of recombinant basic fibroblast growth factor, insulin-like growth factor-II and transforming growth factor-beta 1 on dog dental pulp cells in vivo. Arch Oral Biol. 1998;43:431-444.
[108] Hu CC, Zhang C, Qian Q. Reparative dentin formation in rat molars after direct pulp capping with growth factors. J Endod. 1998;24:744-751.
[109] 杨富生,等。猴前牙尖周病模型的x线片和组织病理学对比观察。现代口腔医学杂志,1989;4:223.
[110] Yamasaki M, Kumasawa M, Kohsaka T, Nakamura H, Kameyama Y. Pulp and periapical tissue after experimental pupal exposure in rats. J Endod. 1994;20:13-17.
[111] Yamasaki M, Nakane A, Kumazawa M, Hashioka K, Horiba N, Nakamura H. Endotoxin and Gram negative bacteria in the rat periapical lesion. J Endod. 1992;18:501-504.
[112] 曹峰,赵怡芳,熊卫星,韩新光。 实验性根尖周病变的X线及组织学研究。广东牙病防治,1997;5:7-9.
[113] Siqueira JF, Lima KC, Magalheas F, et al. Mechanical reduction of the bacteria population in the root canal by three instrumentation techniques. J Endod. 1999;25:332-335.
[114] Pacios MG, de la Casa ML, de Bulacio MA, López ME. Influence of different vehicles on the pH of calcium hydroxide pastes. J Oral Sci.2004;46:107-111.
[115] Menendez A, Brett Finlay B. Defensins in the immunology of bacterial infections. Curr Opin Immunol. 2007;19:385-391.
[2] 李建民,周开亚,张双全。先天免疫中的抗菌肽及其进化。南京师大学报(自然科学版),1999;22:16.
[3] Boman HG. Antibacterial peptides: key components needed in immunity. Cell. 1991;65:205-207.
[4] 张继南,陈红霞。抗菌肽及其应用研究进展。 生物技术通讯,2006;17:669-672.
[5] Lehrer RI, Ganz T, Selsted ME. Defensins: endogenous antibiotic peptides of animal cells. Cell. 1991;64:229-230.
[6] Kimbrell DA. Insect antibacterial proteins: not just for insects and against bacteria. Bioessays. 1991;13:657-663.
[7] Hoffmann JA, Hetru C. Insect defensins: inducible antibacterial peptides. Immunol Today. 1992;13:411-415.
[8] Kagan BL, Ganz T, Lehrer RI. Defensins: a family of antimicrobial and cytotoxic peptides. Toxicology. 1994;87(1-3):131-149.
[9] Hultmark D. Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet. 1993;9:178-183.
[10] Bensch KW, Raida M, M?gert HJ, Schulz-Knappe P, Forssmann WG. hBD-1: a novel beta-defensin from human plasma. FEBS Lett. 1995;368:331-335.
[11] 李春丽,郑振宇,赵卫东。人防御素的研究进展及其应用前景。中国生物制品学杂志,2007;20:146-148.
[12] Schroder JM. Epithelial antimicrobial peptides: innate local host response elements. Cell Mol Life Sci. 1999;56:32-46.
[13] Garcia-Olmedo F, Rodriguez-Palenzuela P, Molina A, Alamillo JM, López-Solanilla E, Berrocal-Lobo M, Poza-Carrión C. Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence. FEBS Lett. 2001;498:219-222.
[14] Broekaert WF, Terras FR, Cammue BP, Osborn RW. Plant defensins: novel antimicrobial peptides as components of the host defense system. Plant Physiol. 1995;108:1353-1358.
[15] Broekaert WF, Cammue BPA, De Bolle MFC, et al. Antimicrobial peptides from plants. Crit Rev Plant Sci. 1997;16:297-323.
[16] Terras FR, Torrekens S, Van Leuven F, Osborn RW, Vanderleyden J, Cammue BP, Broekaert WF. A new family of basic cystein-rich antifungal proteins from Brassicaceae species. FEBS Lett. 1993;16:233-240.
[17] Steiner H, Hultmark D, Engstr?m A, Bennich H, Boman HG. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature. 1981;292:246-248.
[18] Fehlbaum P, Bulet P, Michaut L, Lagueux M, Broekaert WF, Hetru C, Hoffmann JA. Insect immunity. Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. J Biol Chem. 1994;269:33159-33163.
[19] Lee JY, Edlund T, Ny T, Faye I, Boman HG. Insect immunity. Isolation of cDNA clones corresponding to attacins and immune protein P4 from Hyalophora cecropia. EMBO J. 1983;2:577-581.
[20] Lamberty M, Zachary D, Lanot R, Bordereau C, Robert A, Hoffmann JA, Bulet P. Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect. J Biol Chem. 2001;276:4085-4092.
[21] Imler JL, Tauszig S, Jouanguy E, Forestier C, Hoffmann JA. LPS-induced immune response in Drosophila. J Endotoxin Res. 2000;6:459-462.
[22] Tang YQ, Yuan J, Osapay G, Osapay K, Tran D, Miller CJ, Ouellette AJ, Selsted ME. A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science. 1999;286:498-502.
[23] Tran D, Tran PA, Tang YQ, Yuan J, Cole T, Selsted ME. Homodimeric theta-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides. J Biol Chem. 2002;277:3079-3084.
[24] Bals R. Epithelial antimicrobial peptides in host defense against infection. Respir Res. 2000;1:141-150.
[25] Boman HG. Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scand J Immunol .1998;48:15-25.
[26] Panyutich AV, Panyutich EA, Krapivin VA, Baturevich EA, Ganz T. Plasma defensin concentrations are elevated in patiens with septicemia and baterial meningitis. J Lab Clin Med. 1993;122:202-207.
[27] van Wetering S, Sterk PJ, Rabe KF, Hiemstra PS. Defensins: key players or bystanders in infection, injury, and repair in the lung. J Allergy Clin Immunol. 1999;104:1131-1138.
[28] Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T, Tack BF, McCray PB Jr. Productions of beta-defensins by human airway epithelia. Proc Natl Acad Sci USA.1998;95:14961-14966.
[29] Lillard JW Jr, Boyaka PN, Chertov O, Oppenheim JJ, McGhee JR. Mechanisms for induction of acquired host immunity by neutrophil peptide defensins. Proc Natl Acad Sci USA. 1999;96:651-656.
[30] van Wetering S, Mannesse-Lazeroms SP, Dijkman JH, Hiemstra PS. Effect of neutrophil serine proteinases and defensins on lung epithelial cells:modulation of cytoxicity and IL-8 production. J Leukoc Biol. 1997;62:217-226.
[31] Diamond G, Zasloff M, Eck H, Brasseur M, Maloy WL, Bevins CL. Tracheal antimicrobial peptide, a novel cystein-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA. Proc Natl Acad Sci USA. 1991;88:3952-3956.
[32] Harder J, Bartels J, Christophers E, Schr?der JM. A peptide antibiotic from human skin. Nature. 1997;387:861.
[33] Schutte BC, Mitros JP, Bartlett JA, Walters JD, Jia HP, Welsh MJ, Casavant TL, McCray PB Jr. Discovery of five conserved beta-defensin gene clusters using a computational search strategy. Proc Natl Acad Sci USA. 2002;99:2129-2133.
[34] Harder J, Siebert R, Zhang Y, Matthiesen P, Christophers E, Schlegelberger B, Schr?der JM. Mapping of the gene encoding human beta-defensin-2 (DEFB2) to chromosome region 8p22-p23.1. Genomics. 1997;46:472-475.
[35] Liu L, Wang L, Jia HP, Zhao C, Heng HH, Schutte BC, McCray PB Jr, Ganz T. Structure and mapping of the human beta-defensin HBD-2 gene and its expression at sites of inflammation. Gene. 1998;222:237-244.
[36] Jia HP, Schutte BC, Schdy A, Linzmeier R, Guthmiller JM, Johnson GK, Tack BF, Mitros JP, Rosenthal A, Ganz T, McCray PB Jr. Discovery of newhuman beta-defensins using a genomics-based approach. Gene. 2001;263:211-218.
[37] Harder J, Bartels J, Christophers E, Schroder JM. Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic. J Biol Chem. 2001;276:5707-5713.
[38] Garcia JR, Krause A, Schulz S, Rodríguez-Jiménez FJ, Klüver E, Adermann K, Forssmann U, Frimpong-Boateng A, Bals R, Forssmann WG. Human beta-defensin 4: a novel inducible peptide with a specific salt-sensitive spectrum of anti-microbial activity. FASEB J. 2001;15:1819-l821.
[39] Oppenheim JJ, Biragyn A, Kwak LW, Yang D. Roles of antimierobial peptides such as defensins in innate and adaptive immunity. Ann Rheum Dis. 2003;62:1117-1121.
[40] Dhople V, Krukemeyer A, Ramamoorthy A. The human beta-defensin-3, an antibacterial peptide with multiple biological functions. Biochimica et Biophysica Acta. 2006;1758:1499-1512.
[41] Valore EV, Park CH, Quayle AJ, Wiles KR, McCray PB Jr, Ganz T. Human beta-defensin-1: an antimicrobial peptide of urogenital tissues. J Clin Invest. 1998;101:1633-1642.
[42] Bals R, Wang X, Wu Z, Freeman T, Bafna V, Zasloff M, Wilson JM. Human beta-defensin 2 is a salt-sensitive peptide antibiotic expressed in human lung. J Clin Invest. 1998;102:874-880.
[43] Diamond G, Jones DE, Bevins CL. Airway epithelial cells are the site of expression of a mammalian antimicrobial peptide gene. Proc Natl Acad Sci USA. 1993;90:4596-4600.
[44] Ganz T. Defensins and host defense. Science. 1999;286:420-421.
[45] Hoover DM, Rajashankar KR, Blumenthal R, Puri A, Oppenheim JJ, Chertov O, Lubkowski J. The structure of human beta-defensin-2 shows evidence of higher order oligomerization. J Biol Chem. 2000;275:32911-32918.
[46] Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schr?der JM, Wang JM, Howard OM, Oppenheim JJ. Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science. 1999;286:525-528.
[47] Solomon S, Hu J, Zhu Q, Belcourt D, Bennett HP, Bateman A, Antakly T. Corticostatic peptides. J Steroid Biochem Mol Biol. 1991;40:391-398.
[48] Becker MN, Diamond G, Verghese MW, Randell SH. CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem. 2000;275:29731-29736.
[49] Hao HN, Zhao J, Lotoczky G, Grever WE, Lyman WD. Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines. J Neurochem. 2001;77:1027-1035.
[50] Abiko Y, Suraweera AK, Nishimura M, Arakawa T, Takuma T, Mizoguchi I, Kaku T. Differential expression of human beta-defensin-2 in keratinized and non-keratinized oral epithelial lesions; imunohis tochemistry and in situ hybridization. Virchows Arch. 2001;438:248-253.
[51] Mizukawa N, Sawaki K, Yamachika E, Fukunaga J, Ueno T, Takagi S, Sugahara T. Presence of human beta-defensin-2 in oral squamous cell carcinoma. Anticancer Res. 2000;20:2005-2007.
[52] Wada A, Mori N, Oishi K, Hojo H, Nakahara Y, Hamanaka Y, Nagashima M, Sekine I, Ogushi K, Niidome T, Nagatake T, Moss J, Hirayama T. Induction of human beta-defensin-2 mRNA expressi on by Helicobacterpylori in human gastric cell line MKN45 cells on cag pathogeni city island. Biochem Biophys Res Commun. 1999;263:770-774.
[53] Abiko Y, Mitamura J, Nishimura M, Muramatsu T, Inoue T, Shimono M, Kaku T. Pattern of expression of beta-defensins in oral squamous cell carcinoma. Cancer Lett. 1999;143:37-43.
[54] Tomita T, Hitomi S, Nagase T, Matsui H, Matsuse T, Kimura S, Ouchi Y. Effect of ions on antibacterial activity of human beta-defensin-2. Microbiol Immunol. 2000;44:749-754.
[55] Duits LA, Rademaker M, Ravensbergen B, van Sterkenburg MA, van Strijen E, Hiemstra PS, Nibbering PH. Inhibition of hBD-3, but not hBD-1 and hBD-2, mRNA expression by corticosteroids. Biochem Biophys Res Commun. 2001;280:522-525.
[56] Ganz T. Defensins and other antimicrobial peptides: a historical perspective and an update. Comb Chem High Throughput Screen. 2005;8:209-217.
[57] Batoni G, Maisetta G, Esin S, Campa M. Human beta-defensin-3: a promising antimicrobial peptide. Mini-Reviews in Medicinal Chemistry. 2006;6:1063-1073.
[58] Schmid P, Grenet O, Medina J, Chibout SD, Osborne C, Cox DA. An intrinsic antibiotic mechanism in wounds and tissue-engineered skin. J Invest Dermatol. 2001;116:471-472.
[59] Kisich KO, Heifets L, Higgins M, Diamond G. Antimycobacterial agent based on mRNA encoding human beta-defensin-2 enables primary macrophages to restrict growth of mycobacterium tuberculosis. Infect Immun. 2001;69:2692~2699.
[60] Schibli DJ, Hunter HN, Aseyev V, Starner TD, Wiencek JM, McCray PB Jr, Tack BF, Vogel HJ. The solution structures of the human β-defensins lead toa better understanding of the potent bactericidal activity of HBD3 against Saureus. J Biol Chem, 2002;277:8279-8289.
[61] Joly S, Organ C, Johnson GK, McCray PB Jr, Guthmiller JM. Correlation between β-defensin expression and induction profiles in gingival keratinocytes. Mol Immunol. 2005;42:1073-1084.
[62] Harder J, Meyer-Hoffert U, Wehkamp K, et a1. Differential gene induction of human β-defensins (hBD1,-2,-3,and-4) in keratinocytes is inhibited by retinoic acid. J Invest Dennatol. 2004;123:522-529.
[63] Kragol G, Lovas S, Varadi G, Condie BA, Hoffmann R, Otvos L Jr. The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry. 2001;40:3016-3026.
[64] Hancock RE, Diamond G. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 2000;8:402-410.
[65] Ganz T. Defensins: antimicrobial peptides of innate immunity. Nat Rev Imnmnol. 2003;3:710-720.
[66] Quinones-Mateu ME, Lederman MM, Feng Z, Chakraborty B, Weber J, Rangel HR, Marotta ML, Mirza M, Jiang B, Kiser P, Medvik K, Sieg SF, Weinberg A. Human epithelial beta-defensins 2 and 3 inhibit HIV-l replication. AIDS. 2003;17:F39-48.
[67] Wang Z, Wang G. APD: the Antimicrobial Peptide Database. Nucleic Acids Res. 2004;32(Database issue):D590-592.
[68] 司利钢,刘玺诚,李文梅,吕有勇,饶小春,马渝燕。大蒜素对肺癌A549细胞株β防御素表达的影响。首都医科大学学报,2006;27:792-794.
[69] Dunsche A, Acil Y, Siebert J, Harder J, Schr?der JM, Jepsen S. Expression profile of human defensins and antimicrobial proteins in oraltissues. J Oral Pathol Med. 2001;30:154-158.
[70] 钟德钰,杨美薷,吴琦,王伯瑶。人中性粒细胞防御素对龈下微生物的影响。华西口腔医学杂志,1997;15:123-125.
[71] Miyasaki KT, Lehrer RI. Beta-sheet antibiotic peptides as potential dental therapeutics. Int J Antimicrob Agents. 1998;9:269-280.
[72] Miyasaki KT, Iofel R, Huynh T, Huynh T, Lehrer RI. Killing of Fusobacterium nucleatum, Prophyromona gingivalis and Prevotella intermedia by protegrins. J Periodont Res. 1998;33:91-98.
[73] 刘进,奚 涛。人β防御素-3的研究进展。药物生物技术. 2005;12:337-341.
[74] 彭力,徐志南,方向明,吴金民; 岑沛霖。人防御索的研究进展。生物工程学报. 2003;19:261-266.
[75] Sahly H, Schubert S, Harder J, Rautenberg P, Ullmann U, Schr?der J, Podschun R. Burkholderia is highly resistant to human Beta-defensin-3. Antimicrob Agents Chemother. 2003;47:1739-1741.
[76] 扈英伟,朱敏,刘正。乳牙感染根管的菌丛分析。上海口腔医学,1998;7:143-146.
[77] Sato T, Hoshino E, Uematsu H, et al. Predominant obligate anaerobes in necrotic pulps of human deciduous teeth. Microbial Ecology in Health and Disease. 1993;6:269-275.
[78] Lu Q, Samaranayake LP, Darveau RP, Jin L. Expression of human beta-defensin-3 in gingival epithelia. J Periodontal Res. 2005;40:474-481.
[79] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning, a laboratory manual. 3nd ed. NewYork: Cold Spring Harbor Laboratory Press, 2002.
[80] 汪 家 政 , 范 明 。 蛋 白 质 技 术 手 册 , 北 京 : 科 学 出 版 社 ,2000;8:82-92;169-178.
[81] Schagger H, Von Jagow G. Tricine-sodium dodecyl sulfatepolyacrylamidegel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166:368-379.
[82] 肖晓蓉。口腔微生物学及实用技术,北京:北京医科大学出版社,1993;12:178;165-167.
[83] Goeddel DV. Systems for heterologous gene expression. Methods Enzymol, 1990;185:3-7.
[84] Balbas P, Bolivar F. Design and construction of expression plasmid vectors in Escherichia coli. Methods Enzymol, 1990;185:14-37.
[85] Hockney RC. Recent developments in heterologous protein production in Escherichia coli. Trends-Biotechnol, 1994;12:456-463.
[86] 卢圣栋。现代分子生物学实验技术。第二版,北京:中国协和医科大学出版社。1999:365-387.
[87] Hughes KT, Maloy SR. Use of operon and gene fusions to study gene regulation in Salmonella. Methods Enzymol. 2007;421:140-158.
[88] Hoover DM, Wu ZB, Tucker K, Lu W, Lubkowski J. Antimierobial characterization of humanβ-denfensin 3 derivatives. Antimlerob Agents Chemother, 2003;47:2804-2809.
[89] Forstner M, Leder L, Mayr LM. Optimization of protein expression systems for modern drug discovery. Expert Rev Proteomics. 2007;4:67-78.
[90] 随广超,胡美浩。影响大肠杆菌外源基因表达的因素。生物化学与生物物理进展。1994;21:128-132.
[91] 陈姗,何风田,董燕麟,李蓉芬,高会广,陈敏,彭家和。人β防御素3融合蛋白在大肠杆菌中的表达、纯化和活性分析。生物工程学报,2004;20:490-495.
[92] Rosa OP, Torres SA, Ferreira CM, Ferreira FB. In vitro effect of intracanal medicaments on strict anaerobes by means of the broth dilution method.Pesqui Odontol Bras. 2002;16:31-36.
[93] 肖晓蓉。口腔微生物学及实用技术,北京:北京医科大学出版社,1993;12:178;160-165.
[94] Gomes BP, Drucker DB, Lilley JD. Associations of specific bacteria with some endodontic signs and symptoms. Int Endod J. 1994;27:291-298.
[95] Gomes BP, Pinheiro ET, Gadê-Neto CR, Sousa EL, Ferraz CC, Zaia AA, Teixeira FB, Souza-Filho FJ. Microbiological examination of infected dental root canals. Oral Microbiol Immunol. 2004;19:71-76.
[96] Yoshida M, Fukushima H, Yamamoto K, Ogawa K, Toda T, Sagawa H. Correlation between clinical symptoms and microorganisms isolated from root canals of teeth with periapical pathosis. J Endod. 1987;13:24-28.
[97] 邓惠妹,赵雪梅。急性根尖周炎患牙根管内厌氧菌的细菌学研究。华西口腔医学杂志,1993;11:148-150.
[98] 梁景平,刘正,张国驰。感染根管厌氧菌的初步研究。中华口腔医学杂志,1991,26:28-30.
[99] Siqueria JF, Goncalves RB. Antibacterial activities of root canal sealers against selected anaerobic bacteria. Journal of Endod. 1996;22:89-90.
[100] Nakajo K, Nakazawa F, Iwaku M, Hoshino E. Alkali-resistant bacteria in root canal systems. Oral Microbiol Immunol. 2004;19:390-394.
[101] 章魁华,于世凤。实验口腔病理学。北京:人民卫生出版社,2002.29-31.
[102] 肖晓蓉。口腔微生物学及实用技术,北京:北京医科大学出版社,1993;12:95-98.
[103] 吉男匡宏 . 新しぃ概念に基づく根管治疗の实际 . 齿界展望 . 2001;97:805-810.
[104] 王伯沄,李玉松,黄高焺,张远强。病理学技术。北京:人民卫生出版社,2000.73
[105] Barbosa SV, Araki K, Spangberg LS. Cytotoxicity of some modified root canal sealers and their leachable components. Oral Surg Oral Med Oral Pathol, 1993;75:357-361.
[106] 张真,卢晓风。生物材料有效性和安全性评价的现状和趋势。生物医学工程杂志,2002;l9:ll7.
[107] Tziafas D, Alvanou A, Papadimitriou S. Effects of recombinant basic fibroblast growth factor, insulin-like growth factor-II and transforming growth factor-beta 1 on dog dental pulp cells in vivo. Arch Oral Biol. 1998;43:431-444.
[108] Hu CC, Zhang C, Qian Q. Reparative dentin formation in rat molars after direct pulp capping with growth factors. J Endod. 1998;24:744-751.
[109] 杨富生,等。猴前牙尖周病模型的x线片和组织病理学对比观察。现代口腔医学杂志,1989;4:223.
[110] Yamasaki M, Kumasawa M, Kohsaka T, Nakamura H, Kameyama Y. Pulp and periapical tissue after experimental pupal exposure in rats. J Endod. 1994;20:13-17.
[111] Yamasaki M, Nakane A, Kumazawa M, Hashioka K, Horiba N, Nakamura H. Endotoxin and Gram negative bacteria in the rat periapical lesion. J Endod. 1992;18:501-504.
[112] 曹峰,赵怡芳,熊卫星,韩新光。 实验性根尖周病变的X线及组织学研究。广东牙病防治,1997;5:7-9.
[113] Siqueira JF, Lima KC, Magalheas F, et al. Mechanical reduction of the bacteria population in the root canal by three instrumentation techniques. J Endod. 1999;25:332-335.
[114] Pacios MG, de la Casa ML, de Bulacio MA, López ME. Influence of different vehicles on the pH of calcium hydroxide pastes. J Oral Sci.2004;46:107-111.
[115] Menendez A, Brett Finlay B. Defensins in the immunology of bacterial infections. Curr Opin Immunol. 2007;19:385-391.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |