抗牙龈卟啉单胞菌卵黄抗体对大鼠实验性牙周炎的治疗作用
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摘要
目的观察并评价特异性抗牙龈卟啉单胞菌卵黄抗体(IgY)体外抑制牙周主要致病菌牙龈卟啉单胞菌生长和减轻牙周组织炎症程度的作用;为免疫防治慢性牙周炎探索新的解决途径。
方法将提出纯化出的抗牙龈卟啉单胞菌-IgY稀释成2mg/ml,1mg/ml,0.5mg/ml,0.25mg/ml,0.125mg/ml和0.0625mg/ml 6个浓度,厌氧环境培养牙龈卟啉单胞菌国际标准菌株ATCC 33277,分别采用微量稀释法和纸片扩散法测定抗牙龈卟啉单胞-IgY的最小抑菌浓度和抑菌圈直径大小,评价抗牙龈卟啉单胞菌-IgY体外抑菌的能力;采用双层丝线结扎加牙龈卟啉单胞菌接种同时辅以高糖饮料的方法在7周内建立大鼠的实验性牙周炎模型,分为抗牙龈卟啉单胞菌-IgY组、米诺环素组、PBS组、模型组和空白对照组5组,待牙周炎模型形成后按实验分组情况分别给予治疗,记录实验期间大鼠体重、一般情况和探诊深度、龈沟出血指数、松动度等牙周临床指标的变化,每3天治疗一次,连续治疗4周后处死动物取出上下颌骨,截取实验牙位进行固定、脱矿、包埋、切片并行HE染色,观察抗牙龈卟啉单胞菌-IgY治疗的实验牙位在组织学上的变化,并比较抗牙龈卟啉单胞菌-IgY和临床常用抗菌药物米诺环素在治疗效果上是否存在差异,评价抗牙龈卟啉单胞菌-IgY抑制慢性牙周炎、改善牙周组织炎症的作用。实验数据采用配对t检验进行统计分析(SPSS 10.0),P<0.05表示存在统计学差异。
结果经比较在不同浓度共培养下的菌液的OD值可测定抗牙龈卟啉单胞菌-IgY的最小抑菌浓度为0.5mg/ml;最大抑菌圈直径可达15.16±0.33mm。在动物实验中,大鼠生命体征平稳,无明显的毒副反应。分组治疗后,抗牙龈卟啉单胞菌-IgY组和PBS对照组的各项牙周临床指标统计结果均存在显著性差异(P<0.05);和米诺环素组的各项牙周临床指标在统计学上无显著性差异(P>0.05),两者在治疗后牙周临床症状均得到明显改善,牙龈色形质逐渐恢复正常,松动度减小,提示了抗牙龈卟啉单胞菌-IgY可取得与米诺环素相似的临床治疗效果;病理学切片染色结果表明经抗牙龈卟啉单胞菌-IgY治疗后,牙周组织的炎症得到了控制并存在一定的组织修复反应。
结论抗牙龈卟啉单胞菌-IgY在体外可获得良好的抑菌效果,用于实验动物口腔内可明显改善实验牙位的牙周炎症,且不会对受试对象产生毒副反应,是一种安全、有效的生物制剂。该种抗体在免疫防治牙周炎方面具有良好的发展前景。
Objective To evaluate the effects of egg yolk immunoglobulin (IgY) against Porphyromonas gingivalis in vitro and reduce the degree of periodontal tissue inflammation in vivo, explore a new immune approach to prevent and cure the chronic periodontitis.
Methods The puried anti-Pg IgY was diluted six different concentration (2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml) and investigated the ihibiton effects of anti-Pg IgY on Pg (ATCC 33277) through discagar diffusion test and dilution test in vitro. Periodontitis was induced by silk ligature technique in male Sprague-Dawley rats, then the rats were fed with 100 g/L high sucrose drinking water and Pg 7 weeks. 15 adult male Sprague-Dawley rats were divided into 5 groups:①Rats with periodontitis treated by anti-Pg IgY (2mg/ml),②Rats with periodontitis treated by minocycline hydrochloride (2mg/ml),③R ats with periodontitis treated by equal volum of PBS,④periodontitis control and⑤the blank control. We recorded the weight of rats, mobility (MOB) of periodontitis, probing depth (PD) and sulcus bleeding index (SBI). After four-week-treatment, the tissues around the test site were excised, samples were embedded and fixed, then the histological assessment were carried out by HE staining to evaluate whether the test sites treated by anti-Pg IgY than before. Then compared the groups treat with anti-Pg IgY and minocycline hydrochloride. Differences between experimental groups were tested with paried t-text, using SPSS 10.0. P values less than 0.05 were considered significant. The animal experiment will evaluate anti-Pg IgY inhibition effects on periodontal tissue inflammation in vivo.
Results Compared the OD value, we found the minimal inhibitory concentration of anti-Pg IgY is 0.5mg/ml, and the maxium diameter of bacteriostasis-collares is 15.16±0.33mm. During the vivo study, there were no obvious changes in the weight of rats of any groups and no symptom of toxicity were observed. The periodontitis control rats had bacterial plaque assembled on gingival sulcus inner margin epithelium. Pathology examination also showed a significant gingival epithlium anabrosis and a notable inflammatory cells infiltration. Parts of periodontal membrane spaces became wide, angiectasis and engorge, the alveolar bone was destroyed and absorbed. Anti-Pg IgY and minocycline hydrochloride could significantly improved the periodontal clinical paraments, and there was no significantly difference between them (P>0.05). Anti-Pg IgY and minocycline hydrochloride could lighten the periodontal inflammation, reduced the probing depth and the mobility of test site; However, there are significantly difference between anti-Pg IgY and PBS group (P<0.05). The pathology examination of HE staining also indicated that the test sites which were treated with anti-Pg IgY have a certainly control of gingival inflammation, and the tissue could repaired. These data means anti-Pg IgY could achieve the similar clinic therapeutics with minocycline hydrochloride.
Conclusion Our study showed that anti-Pg IgY is kind of effectively immunotherapeutic agent without toxicity, it could effectivly inhibitied Pg in vitro and improved the periodontal tissue inflammation in rats. Anti-Pg IgY have and a bright application foreground in periodontitis immune prophylaxis and treatment.
方法将提出纯化出的抗牙龈卟啉单胞菌-IgY稀释成2mg/ml,1mg/ml,0.5mg/ml,0.25mg/ml,0.125mg/ml和0.0625mg/ml 6个浓度,厌氧环境培养牙龈卟啉单胞菌国际标准菌株ATCC 33277,分别采用微量稀释法和纸片扩散法测定抗牙龈卟啉单胞-IgY的最小抑菌浓度和抑菌圈直径大小,评价抗牙龈卟啉单胞菌-IgY体外抑菌的能力;采用双层丝线结扎加牙龈卟啉单胞菌接种同时辅以高糖饮料的方法在7周内建立大鼠的实验性牙周炎模型,分为抗牙龈卟啉单胞菌-IgY组、米诺环素组、PBS组、模型组和空白对照组5组,待牙周炎模型形成后按实验分组情况分别给予治疗,记录实验期间大鼠体重、一般情况和探诊深度、龈沟出血指数、松动度等牙周临床指标的变化,每3天治疗一次,连续治疗4周后处死动物取出上下颌骨,截取实验牙位进行固定、脱矿、包埋、切片并行HE染色,观察抗牙龈卟啉单胞菌-IgY治疗的实验牙位在组织学上的变化,并比较抗牙龈卟啉单胞菌-IgY和临床常用抗菌药物米诺环素在治疗效果上是否存在差异,评价抗牙龈卟啉单胞菌-IgY抑制慢性牙周炎、改善牙周组织炎症的作用。实验数据采用配对t检验进行统计分析(SPSS 10.0),P<0.05表示存在统计学差异。
结果经比较在不同浓度共培养下的菌液的OD值可测定抗牙龈卟啉单胞菌-IgY的最小抑菌浓度为0.5mg/ml;最大抑菌圈直径可达15.16±0.33mm。在动物实验中,大鼠生命体征平稳,无明显的毒副反应。分组治疗后,抗牙龈卟啉单胞菌-IgY组和PBS对照组的各项牙周临床指标统计结果均存在显著性差异(P<0.05);和米诺环素组的各项牙周临床指标在统计学上无显著性差异(P>0.05),两者在治疗后牙周临床症状均得到明显改善,牙龈色形质逐渐恢复正常,松动度减小,提示了抗牙龈卟啉单胞菌-IgY可取得与米诺环素相似的临床治疗效果;病理学切片染色结果表明经抗牙龈卟啉单胞菌-IgY治疗后,牙周组织的炎症得到了控制并存在一定的组织修复反应。
结论抗牙龈卟啉单胞菌-IgY在体外可获得良好的抑菌效果,用于实验动物口腔内可明显改善实验牙位的牙周炎症,且不会对受试对象产生毒副反应,是一种安全、有效的生物制剂。该种抗体在免疫防治牙周炎方面具有良好的发展前景。
Objective To evaluate the effects of egg yolk immunoglobulin (IgY) against Porphyromonas gingivalis in vitro and reduce the degree of periodontal tissue inflammation in vivo, explore a new immune approach to prevent and cure the chronic periodontitis.
Methods The puried anti-Pg IgY was diluted six different concentration (2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml) and investigated the ihibiton effects of anti-Pg IgY on Pg (ATCC 33277) through discagar diffusion test and dilution test in vitro. Periodontitis was induced by silk ligature technique in male Sprague-Dawley rats, then the rats were fed with 100 g/L high sucrose drinking water and Pg 7 weeks. 15 adult male Sprague-Dawley rats were divided into 5 groups:①Rats with periodontitis treated by anti-Pg IgY (2mg/ml),②Rats with periodontitis treated by minocycline hydrochloride (2mg/ml),③R ats with periodontitis treated by equal volum of PBS,④periodontitis control and⑤the blank control. We recorded the weight of rats, mobility (MOB) of periodontitis, probing depth (PD) and sulcus bleeding index (SBI). After four-week-treatment, the tissues around the test site were excised, samples were embedded and fixed, then the histological assessment were carried out by HE staining to evaluate whether the test sites treated by anti-Pg IgY than before. Then compared the groups treat with anti-Pg IgY and minocycline hydrochloride. Differences between experimental groups were tested with paried t-text, using SPSS 10.0. P values less than 0.05 were considered significant. The animal experiment will evaluate anti-Pg IgY inhibition effects on periodontal tissue inflammation in vivo.
Results Compared the OD value, we found the minimal inhibitory concentration of anti-Pg IgY is 0.5mg/ml, and the maxium diameter of bacteriostasis-collares is 15.16±0.33mm. During the vivo study, there were no obvious changes in the weight of rats of any groups and no symptom of toxicity were observed. The periodontitis control rats had bacterial plaque assembled on gingival sulcus inner margin epithelium. Pathology examination also showed a significant gingival epithlium anabrosis and a notable inflammatory cells infiltration. Parts of periodontal membrane spaces became wide, angiectasis and engorge, the alveolar bone was destroyed and absorbed. Anti-Pg IgY and minocycline hydrochloride could significantly improved the periodontal clinical paraments, and there was no significantly difference between them (P>0.05). Anti-Pg IgY and minocycline hydrochloride could lighten the periodontal inflammation, reduced the probing depth and the mobility of test site; However, there are significantly difference between anti-Pg IgY and PBS group (P<0.05). The pathology examination of HE staining also indicated that the test sites which were treated with anti-Pg IgY have a certainly control of gingival inflammation, and the tissue could repaired. These data means anti-Pg IgY could achieve the similar clinic therapeutics with minocycline hydrochloride.
Conclusion Our study showed that anti-Pg IgY is kind of effectively immunotherapeutic agent without toxicity, it could effectivly inhibitied Pg in vitro and improved the periodontal tissue inflammation in rats. Anti-Pg IgY have and a bright application foreground in periodontitis immune prophylaxis and treatment.
引文
1.周婷,徐屹,丁一,等.慢性牙周炎龈下菌斑中五种牙周可疑致病微生物的分布[J].华西口腔医学杂志,2007,25(5): 470-473.
2.Akiyo KI, Kazuyuki I, Sachiyo T, et al. Investigation of subgingival profile of periodontopathic bacteria using Polymerase Chain Reaction. [J] Bull Tokyo Dent Coll, 2010, 51(3): 139-144.
3.Masunaqa H, Tsutae W, Oh H, et al. Use of quantitative PCR to evaluate methods of bacteria sampling in periodontal patients [J]. J Oral Sci, 2010, 52(4): 615-621.
4.Bullon P, Cordero MD, Quiles JL, et al. Mitochondrial dysfunction promoted by Porphyromonas gingivalis lipopolysaccharide as a possible link between cardiovascular disease and periodontitis [J]. Free Radic Biol Med, 2011, Feb 24. [Epub ahead of print].
5 . Hayashi C, Viereck J, Hua N, et al. Porphyromonas gingivalis accelerates inflammatory atherosclerosis in the innominate artery of ApoE deficient mice [J]. Atherosclerosis. 2011, 215(1): 52-59.
6.Yamazaki K, Honda T, Domon H, et al. Relationship of periodontal infection to serum antibody levels to periodontopathic bacteria and inflammatory markers in periodontitis patients with coronary heart disease [J]. Clin Exp Immunol, 2007, 149(3): 445-452.
7.Tonetti MS, D’Aiuto F, Nibali L, et al. Treatment of periodontitis and endothelial function[J]. New Engl J Med,2007,35(6): 911-920.
8.Koichiro W, Yoshinori K. Roles of oral bacteria in Cardiovascular disease-from molecular mechanisms to clinical cases: involvement of Porphyromonas gingivalis in the development of human aortic aneurysm [J]. J Pharmacol Sci, 2010, 11(3):115-119.
9.宋红,马秦,吕昕,等.牙龈卟啉单胞菌对心血管内皮细胞黏附和侵入的研究[J].心脏杂志,2010, 22(5): 692-694.
10.Nakano K, Inaba H, Nomura R, et al. Distribution of Porphyromonas gingivals fimA genotypes in cardiovascular specinens from Japanese patients[J]. Oral Microbiol Immunol, 2008, 23(2): 170-172.
11.Haffajee AD, Socransky SS. Microbial etiological agents of destructive preiodontal diseases [J]. Periodontol 2000, 1994, 5(2): 78-111.
12.Frazer LT, O’Brien-Simposon NM, Slakeski N, et al. Vaccination with recombinant adhesins from the RgpA-Kgp proteinase-adhesin complex protects against Porphyromonas gingivalis infection [J]. Vaccine, 2006, 24(42-43): 6542-6554.
13.Fujibayashi T, Nakamura M, Tominaga A, et al. Effects of IgY against Candida albicans and Candida spp. Adherence and Biofilm formation [J]. Jpn J Infect Dis, 2009, 62(5): 337-342.
14.Mujo K, Shinji H, Yoshitomo I, et al. Egg Yolk Antibody and Its Application [J]. Biotechnol Bioprocess Eng, 2000,5(3):79-82.
15.龚晨睿,潘思轶.特异性鸡蛋黄抗体的毒理学安全性研究[J]。食品科学,2006, 27(1):226-230.
16.江千舟,樊明文,李宇红,等.鸡蛋黄抗体IgY的急、慢性毒性实验[J]。牙体牙髓牙周病学杂志,2001,11(6): 38-39.
17.胡国柱,聂荣庆,张进,等.抗牙龈卟啉单胞菌IgY的制备及抑菌效果[J]。实用口腔医学杂志,2010, 26(3): 337-340.
18.戴万明,聂琳。1627名学龄前儿童IgY口腔喷剂防龋效果观察[J]。浙江预防医学,2008,20(11):56-58.
19.王祝玲,王强。抗龋IgY牙膏对乳牙防龋效果的临床研究[J]。牙体牙髓牙周病学杂志,2008,18(6):33-34.
20.曹立群,苏剑生,张懿。IgY牙膏防龋的临床效果观察[J]。同济大学学报(医学版), 2006, 27(3):45-47。
21.Yokoyama K, Sugano N, Shimada T, et al. Effects of egg yolk antibody againstporphyromonas gingivalis gingipains in periodontitis patients [J]. Oral Sci, 2007, 49(3): 201-206.
22.Takahshi N, Eisenhuth G, Lee I, et al. Immunoglobulins in milk from cows immunized with oral strains of Actinomyces, Prevotella, Porphyromonas, and Fusobacterium [J]. J Dent Res, 1992, 71(8): 1509-1515.
23.曹采方主编。牙周病学[M].第2版.北京:人民卫生出版社,2000.
24.Le H, Silness J. Periodontal disease in pregnancy.1. Prevalence and severity [J]. Acta Odontol Scand,1963, 21(5): 533-511.
25.Tanaka T, Kido MA, Ibuki T, et al. Immunocytochemical study of nerve fibers containing substance P in the junctional epithelium of rats [J]. J Periodontal Res, 1996, 31(3): 187-194.
26.刘静波,潘亚萍,俞宁,等.牙龈卟啉单孢菌毒力岛基因检出与慢性牙周炎的相关性研究[J].上海口腔医学,2010,19(4):341-345.
27.Potempa J, Sroka A, Imamura T, et al. Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of multidomain protein complexes [J]. Curr Protein Pept Sci, 2003, 4(6): 397-407.
28. Sojar HT, Genco RJ. Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae [J]. FEMS Immunol Med Microbiol, 2005, 45(1): 25-30.
29 . Rautemaa R, Jarvensivu A, Kari K, et al. Intracellular localization of Porphyromonas gingivalis thiolproteinase in periodontal tissues of chronic periodontitis patients [J]. Ora Dis, 2004, 10(5): 298 -305.
30.Scheres N, Laine ML, Sipos PM, et al. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis [J]. J Periodontal Res. 2011 Feb 17. [Epub ahead of print]
31.Hajishengallis G, Sojar H, Genco RJ, et al. Intracellular signaling and cytokineinduction upon interactions of Prophyromonas gingivalis fimbria with pattern-recognition receptors [J]. Immunol Invest, 2004, 33(2): 157-172.
32.段星宇,孙拓祺,岗桥畅夫,等.牙龈卟啉单孢菌菌毛蛋白A参与诱导小鼠牙槽骨吸收[J]。国际口腔医学杂志,2010,37(3):249-251.
33.张凤秋,杨连甲,吴织芬.牙龈卟啉菌蛋白酶的结构和作用[J]。牙体牙髓牙周病学杂志,2004,14(6):352-354.
34.O-Brien-Simpson NM, Veith PD, Dashper SG, et al. Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire [J]. Curr Protein Pept Sci, 2003, 4(6): 409-426.
35.李琛,刘静波,潘亚萍,等.不同牙周状态下龈下菌斑中牙龈卟啉单孢菌Kgp基因差异研究[J].口腔医学研究,2010,26(1):26-28.
36.Bainbridge BW, Darveau RP. Porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition recePgtor ligand for the innate host defense system [J]. Acta Odontol Scand, 2001, 59(3): 131-138.
37.Wang YH, Jiang J, Zhu Q , et al. Porphyromonas gingivalis lipids inhibit osteoblastic differentiation and function [J]. Infec Immun, 2010, 78(9): 3726-3735.
38.Wang PL, Ohura K, Fujii T, et al. DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues [J]. Biochem Biophys Res Commun, 2003, 305(4): 970-973.
39.王惠宁,曹金芳,余杰.牙龈卟啉单孢菌表面相关物质对T淋巴细胞凋亡的影响[J].口腔医学,2010,30(5):268-271.
40.Chen LL, Wu YM, Yan J, et al. Association between coinfection of porphyromonas gingivalis, actinobacill us actinomycetemcomitans and treponema denticola and periodiontal tissue destrucionin [J]. Chronic Periodontitis. ChinMed,2005,118(11): 9-15.
41.DongD, Liu H, Xiao Q, et al. Affinity purification of egg yolk immunoglobulins IgYwith a stable synthetic ligand [J]. ChromatogrB Analyt Technol Biomed Life Sci, 2008, 8(70): 51-54.
42.Hatta H, Tsuda K, Ozeki M, et al. Passive immunization against dental plaque formation in humans:effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans [J]. Caries Res. 1997, 31(4): 268-274.
43.Attallah AM, Abbas AT, Ismail H, et al. Efficacy of passive immunization with IgY antibodies to a 58-kDa H. pylori antigen on severe gastritis in BALB/c mouse model [J]. J immunoassay Immunochem, 2009, 30(4): 359-377.
44.Sarker SA, Casswall TH, Juneja LR, et al. Randomized, placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea [J]. Pediatr Gastroenterol Nutr, 2001, 32(1): 19-25.
45.黄文学,樊明文,江千舟,等.特异性鸡蛋黄抗体(IgY)对变形链球菌生长及粘附的影响[J]。口腔医学纵横,2002, 18(2):82-83.
46.陈新宇,陈洪,张岚,等.抗龋IgY和IgY牙膏对体外培养变形链球菌影响的电镜观察[J]。牙体牙髓牙周病学杂志,2004, 14(2):98-100.
47.徐叔云,卞如濂,陈修.药理学实验方法[M].第三版,北京:人民卫生出版社,2001.
1. Booth V, Lehner T. Characterisation of a monoclonal antibody and its application in preventing recolonisation by Porphyromonas gingivalis in patients with periodontitis [J]. Periodontal Res. 1997, 32(1): 54-60.
2. Frazer LT, O’Brien-Simposon NM, Slakeski N, et al. Vaccination with recombinant adhesins from the RgpA-Kgp proteinase-adhesin complex protects against Porphyromonas gingivalis infection [J]. Vaccine, 2006,24(42-43): 6542-54.
3. Kazuyuki H, Hideyuki A, Koji U, et al. Passive oral immunization by egg yolk immunoglobulin(IgY) to vibrio cholerae effectively prevents cholera [J]. Acta Med, Okayama, 2010, 64(3): 163-170.
4. Zhang XY, Kurth A, Pauly D, et al. Application of high-titred IgY antibodies in orthopox virus diagnostics [J]. J Chin Pharm Sci, 2008, 17(3): 183-191.
5. Pauly D, Dorner M, Zhang XY, et al. Immunization of chickens with toxins during two years. Monitoring of laying capacity, IgY-concentration and antibody titer development in chickens immunized with ricin and botulinum toxins over a two years period [J]. Poult Sci, 2009,88(2): 281-290.
6.田刚,汪咏新,廖洪利.卵黄免疫球蛋白(IgY)特性、制备及应用[J].临床检验杂志,2009,27(5):398-399.
7. Diasda SW, Tambourqi DV. IgY: A promising antibody for use in immunodiagnostic and in immunotherapy [J]. Vet immunol immunopatholo, 2010, 135(3-4): 173-180.
8.任平国,徐启红.鸡卵黄免疫球蛋白(IgY)分离纯化的研究[J]。现代食品科技,2007,11(23): 24-26.
9. Svendsent L, Crowley A, Ostergaard LH, et al. Development and comparison of purification strategies for chicken antibodies from egg yolk [J]. Lab Anim Sci, 1995,45(1): 89-93.
10. Ko KY, Ahn DU. Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography[J]. Poult Sci, 2007, 86(2): 400-407.
11.薛海燕,宋宏新.鸡卵黄免疫球蛋白分离纯化方法的研究进展[J].陕西科技大学学报,2003,21(4):86-88.
12. Akita EM, Nakai S. Immunoglobulins from egg yolk: isolation and purification[J]. Journal of food science, 2005, 157(3): 629-634.
13.丁建坤,赵文锋,吴梧桐.卵黄抗体技术应用于大规模制备治疗性抗体的研究进展[J].药物生物技术,2009,16(6):590-594.
14. Taylor AI, Fabiane SM, Sutton BJ, Calvert RA. The crystal structure of an avian IgY-Fc fragment reveals conservation with both mammalian IgG and IgE [J]. Biochemistry,2009, 48(3): 558-562.
15.韩水仲,齐宝珠,赵津子,等.卵黄抗体在医药领域中应用的新趋势[J].中国兽医科学,2010,40(10):1093-1097.
16. Sunwoo HH, Wang WW, Sim JS. Detection of Escherichia coli O157: H 7 using chicken immunoglobulin Y [J]. Immunol Lett, 2006,106(2): 191-193.
17. Vasconcellos FA, Coutinho ML, Dasilva EF, et al. Testing different antigen capture ELISA formats for detection of Leptospira spp in human blood serum [J]. Trans R Soc Trop Med Hyg, 2009, 10(16): 1-6.
18. Shimitzu M, Nagashima H, Hashimoto K, et al. Egg yolk antibody(IgY) stability in aqueous solution with high sugar concentrations [J]. Food sci,1994, 59(3): 763-772.
19. Chang HM, Ou-Yang RF, Chen YT, et al. Productivity and some properties of immunoglobulin specific against Streptococcus mutans serotype c in chicken egg yolk (IgY) [J]. J Agric Food Chem, 1999, 47(1): 61-66.
20. Lee KA, Chang SK, Lee YJ, et al. Acid stability of anti-Helicobacter pyroli IgY in aqueous polyol solution [J]. J Biochem Mol Biol, 2002, 35(5): 488-493.
21.陈阿琴,杨志刚,俞颂东.卵黄免疫球蛋白及其应用研究进展[J].中国兽药杂志,2005,39(3):28-31.
22.李春辉,埚立君,俞颂东.卵黄免疫球蛋白及其应用研究进展[J].中国生物制品学杂志,2005,25(1):45-46.
23.戴万明,聂琳. 1627名学龄前儿童IgY口腔喷剂防龋效果观察[J].浙江预防医学,2008,20(11):56-58.
24.王祝玲,王强.抗龋IgY牙膏对乳牙防龋效果的临床研究[J].牙体牙髓牙周病学杂志,2008,18(6):33-34.
25. Fujibayashi T,Nakamura M,Tominaga A, et al. Effects of IgY against Candida albicans and Candida spp. Adherence and Biofilm Formation [J]. Jpn. J. Infect Dis, 2009, 62(5): 337-342.
26.胡国柱,聂荣庆,张进,等.抗牙龈卟啉单胞菌IgY的制备及抑菌效果[J].实用口腔医学杂志,2010, 26(3): 337-340.
27. Okuda K, Kato T, Naito Y. Protective efficacy of active and passive immunizations against experimental infection with Bacteroids gingivalis in ligated hamsters [J]. J Pent Res, 1988, 67(5): 807-811.
28. Takahshi N, Eisenhuth G, Lee I, et al. Immunoglobulins in milk from cows immunized with oral strains of Actinomyces, Prevotella, Porphyromonas, and Fusobacterium [J]. J Dent Res, 1992, 71(8): 1509-1515.
29. Yokoyama K, Sugano N, Rahman AK, et al. Activity of anti-Porphyromonas gingivalis egg yolk antibody against gingipains in vitro [J]. Oral Microbiol Immunol, 2007, 22(5): 352-355.
30. Yokoyama K, Sugano N, Shimada T, et al. Effects of egg yolk antibody against porphyromonas gingivalis gingipains in periodontitis patients [J]. J Oral Sci, 2007, 49(3): 201-206.
2.Akiyo KI, Kazuyuki I, Sachiyo T, et al. Investigation of subgingival profile of periodontopathic bacteria using Polymerase Chain Reaction. [J] Bull Tokyo Dent Coll, 2010, 51(3): 139-144.
3.Masunaqa H, Tsutae W, Oh H, et al. Use of quantitative PCR to evaluate methods of bacteria sampling in periodontal patients [J]. J Oral Sci, 2010, 52(4): 615-621.
4.Bullon P, Cordero MD, Quiles JL, et al. Mitochondrial dysfunction promoted by Porphyromonas gingivalis lipopolysaccharide as a possible link between cardiovascular disease and periodontitis [J]. Free Radic Biol Med, 2011, Feb 24. [Epub ahead of print].
5 . Hayashi C, Viereck J, Hua N, et al. Porphyromonas gingivalis accelerates inflammatory atherosclerosis in the innominate artery of ApoE deficient mice [J]. Atherosclerosis. 2011, 215(1): 52-59.
6.Yamazaki K, Honda T, Domon H, et al. Relationship of periodontal infection to serum antibody levels to periodontopathic bacteria and inflammatory markers in periodontitis patients with coronary heart disease [J]. Clin Exp Immunol, 2007, 149(3): 445-452.
7.Tonetti MS, D’Aiuto F, Nibali L, et al. Treatment of periodontitis and endothelial function[J]. New Engl J Med,2007,35(6): 911-920.
8.Koichiro W, Yoshinori K. Roles of oral bacteria in Cardiovascular disease-from molecular mechanisms to clinical cases: involvement of Porphyromonas gingivalis in the development of human aortic aneurysm [J]. J Pharmacol Sci, 2010, 11(3):115-119.
9.宋红,马秦,吕昕,等.牙龈卟啉单胞菌对心血管内皮细胞黏附和侵入的研究[J].心脏杂志,2010, 22(5): 692-694.
10.Nakano K, Inaba H, Nomura R, et al. Distribution of Porphyromonas gingivals fimA genotypes in cardiovascular specinens from Japanese patients[J]. Oral Microbiol Immunol, 2008, 23(2): 170-172.
11.Haffajee AD, Socransky SS. Microbial etiological agents of destructive preiodontal diseases [J]. Periodontol 2000, 1994, 5(2): 78-111.
12.Frazer LT, O’Brien-Simposon NM, Slakeski N, et al. Vaccination with recombinant adhesins from the RgpA-Kgp proteinase-adhesin complex protects against Porphyromonas gingivalis infection [J]. Vaccine, 2006, 24(42-43): 6542-6554.
13.Fujibayashi T, Nakamura M, Tominaga A, et al. Effects of IgY against Candida albicans and Candida spp. Adherence and Biofilm formation [J]. Jpn J Infect Dis, 2009, 62(5): 337-342.
14.Mujo K, Shinji H, Yoshitomo I, et al. Egg Yolk Antibody and Its Application [J]. Biotechnol Bioprocess Eng, 2000,5(3):79-82.
15.龚晨睿,潘思轶.特异性鸡蛋黄抗体的毒理学安全性研究[J]。食品科学,2006, 27(1):226-230.
16.江千舟,樊明文,李宇红,等.鸡蛋黄抗体IgY的急、慢性毒性实验[J]。牙体牙髓牙周病学杂志,2001,11(6): 38-39.
17.胡国柱,聂荣庆,张进,等.抗牙龈卟啉单胞菌IgY的制备及抑菌效果[J]。实用口腔医学杂志,2010, 26(3): 337-340.
18.戴万明,聂琳。1627名学龄前儿童IgY口腔喷剂防龋效果观察[J]。浙江预防医学,2008,20(11):56-58.
19.王祝玲,王强。抗龋IgY牙膏对乳牙防龋效果的临床研究[J]。牙体牙髓牙周病学杂志,2008,18(6):33-34.
20.曹立群,苏剑生,张懿。IgY牙膏防龋的临床效果观察[J]。同济大学学报(医学版), 2006, 27(3):45-47。
21.Yokoyama K, Sugano N, Shimada T, et al. Effects of egg yolk antibody againstporphyromonas gingivalis gingipains in periodontitis patients [J]. Oral Sci, 2007, 49(3): 201-206.
22.Takahshi N, Eisenhuth G, Lee I, et al. Immunoglobulins in milk from cows immunized with oral strains of Actinomyces, Prevotella, Porphyromonas, and Fusobacterium [J]. J Dent Res, 1992, 71(8): 1509-1515.
23.曹采方主编。牙周病学[M].第2版.北京:人民卫生出版社,2000.
24.Le H, Silness J. Periodontal disease in pregnancy.1. Prevalence and severity [J]. Acta Odontol Scand,1963, 21(5): 533-511.
25.Tanaka T, Kido MA, Ibuki T, et al. Immunocytochemical study of nerve fibers containing substance P in the junctional epithelium of rats [J]. J Periodontal Res, 1996, 31(3): 187-194.
26.刘静波,潘亚萍,俞宁,等.牙龈卟啉单孢菌毒力岛基因检出与慢性牙周炎的相关性研究[J].上海口腔医学,2010,19(4):341-345.
27.Potempa J, Sroka A, Imamura T, et al. Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of multidomain protein complexes [J]. Curr Protein Pept Sci, 2003, 4(6): 397-407.
28. Sojar HT, Genco RJ. Identification of glyceraldehyde-3-phosphate dehydrogenase of epithelial cells as a second molecule that binds to Porphyromonas gingivalis fimbriae [J]. FEMS Immunol Med Microbiol, 2005, 45(1): 25-30.
29 . Rautemaa R, Jarvensivu A, Kari K, et al. Intracellular localization of Porphyromonas gingivalis thiolproteinase in periodontal tissues of chronic periodontitis patients [J]. Ora Dis, 2004, 10(5): 298 -305.
30.Scheres N, Laine ML, Sipos PM, et al. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis [J]. J Periodontal Res. 2011 Feb 17. [Epub ahead of print]
31.Hajishengallis G, Sojar H, Genco RJ, et al. Intracellular signaling and cytokineinduction upon interactions of Prophyromonas gingivalis fimbria with pattern-recognition receptors [J]. Immunol Invest, 2004, 33(2): 157-172.
32.段星宇,孙拓祺,岗桥畅夫,等.牙龈卟啉单孢菌菌毛蛋白A参与诱导小鼠牙槽骨吸收[J]。国际口腔医学杂志,2010,37(3):249-251.
33.张凤秋,杨连甲,吴织芬.牙龈卟啉菌蛋白酶的结构和作用[J]。牙体牙髓牙周病学杂志,2004,14(6):352-354.
34.O-Brien-Simpson NM, Veith PD, Dashper SG, et al. Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire [J]. Curr Protein Pept Sci, 2003, 4(6): 409-426.
35.李琛,刘静波,潘亚萍,等.不同牙周状态下龈下菌斑中牙龈卟啉单孢菌Kgp基因差异研究[J].口腔医学研究,2010,26(1):26-28.
36.Bainbridge BW, Darveau RP. Porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition recePgtor ligand for the innate host defense system [J]. Acta Odontol Scand, 2001, 59(3): 131-138.
37.Wang YH, Jiang J, Zhu Q , et al. Porphyromonas gingivalis lipids inhibit osteoblastic differentiation and function [J]. Infec Immun, 2010, 78(9): 3726-3735.
38.Wang PL, Ohura K, Fujii T, et al. DNA microarray analysis of human gingival fibroblasts from healthy and inflammatory gingival tissues [J]. Biochem Biophys Res Commun, 2003, 305(4): 970-973.
39.王惠宁,曹金芳,余杰.牙龈卟啉单孢菌表面相关物质对T淋巴细胞凋亡的影响[J].口腔医学,2010,30(5):268-271.
40.Chen LL, Wu YM, Yan J, et al. Association between coinfection of porphyromonas gingivalis, actinobacill us actinomycetemcomitans and treponema denticola and periodiontal tissue destrucionin [J]. Chronic Periodontitis. ChinMed,2005,118(11): 9-15.
41.DongD, Liu H, Xiao Q, et al. Affinity purification of egg yolk immunoglobulins IgYwith a stable synthetic ligand [J]. ChromatogrB Analyt Technol Biomed Life Sci, 2008, 8(70): 51-54.
42.Hatta H, Tsuda K, Ozeki M, et al. Passive immunization against dental plaque formation in humans:effect of a mouth rinse containing egg yolk antibodies (IgY) specific to Streptococcus mutans [J]. Caries Res. 1997, 31(4): 268-274.
43.Attallah AM, Abbas AT, Ismail H, et al. Efficacy of passive immunization with IgY antibodies to a 58-kDa H. pylori antigen on severe gastritis in BALB/c mouse model [J]. J immunoassay Immunochem, 2009, 30(4): 359-377.
44.Sarker SA, Casswall TH, Juneja LR, et al. Randomized, placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea [J]. Pediatr Gastroenterol Nutr, 2001, 32(1): 19-25.
45.黄文学,樊明文,江千舟,等.特异性鸡蛋黄抗体(IgY)对变形链球菌生长及粘附的影响[J]。口腔医学纵横,2002, 18(2):82-83.
46.陈新宇,陈洪,张岚,等.抗龋IgY和IgY牙膏对体外培养变形链球菌影响的电镜观察[J]。牙体牙髓牙周病学杂志,2004, 14(2):98-100.
47.徐叔云,卞如濂,陈修.药理学实验方法[M].第三版,北京:人民卫生出版社,2001.
1. Booth V, Lehner T. Characterisation of a monoclonal antibody and its application in preventing recolonisation by Porphyromonas gingivalis in patients with periodontitis [J]. Periodontal Res. 1997, 32(1): 54-60.
2. Frazer LT, O’Brien-Simposon NM, Slakeski N, et al. Vaccination with recombinant adhesins from the RgpA-Kgp proteinase-adhesin complex protects against Porphyromonas gingivalis infection [J]. Vaccine, 2006,24(42-43): 6542-54.
3. Kazuyuki H, Hideyuki A, Koji U, et al. Passive oral immunization by egg yolk immunoglobulin(IgY) to vibrio cholerae effectively prevents cholera [J]. Acta Med, Okayama, 2010, 64(3): 163-170.
4. Zhang XY, Kurth A, Pauly D, et al. Application of high-titred IgY antibodies in orthopox virus diagnostics [J]. J Chin Pharm Sci, 2008, 17(3): 183-191.
5. Pauly D, Dorner M, Zhang XY, et al. Immunization of chickens with toxins during two years. Monitoring of laying capacity, IgY-concentration and antibody titer development in chickens immunized with ricin and botulinum toxins over a two years period [J]. Poult Sci, 2009,88(2): 281-290.
6.田刚,汪咏新,廖洪利.卵黄免疫球蛋白(IgY)特性、制备及应用[J].临床检验杂志,2009,27(5):398-399.
7. Diasda SW, Tambourqi DV. IgY: A promising antibody for use in immunodiagnostic and in immunotherapy [J]. Vet immunol immunopatholo, 2010, 135(3-4): 173-180.
8.任平国,徐启红.鸡卵黄免疫球蛋白(IgY)分离纯化的研究[J]。现代食品科技,2007,11(23): 24-26.
9. Svendsent L, Crowley A, Ostergaard LH, et al. Development and comparison of purification strategies for chicken antibodies from egg yolk [J]. Lab Anim Sci, 1995,45(1): 89-93.
10. Ko KY, Ahn DU. Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography[J]. Poult Sci, 2007, 86(2): 400-407.
11.薛海燕,宋宏新.鸡卵黄免疫球蛋白分离纯化方法的研究进展[J].陕西科技大学学报,2003,21(4):86-88.
12. Akita EM, Nakai S. Immunoglobulins from egg yolk: isolation and purification[J]. Journal of food science, 2005, 157(3): 629-634.
13.丁建坤,赵文锋,吴梧桐.卵黄抗体技术应用于大规模制备治疗性抗体的研究进展[J].药物生物技术,2009,16(6):590-594.
14. Taylor AI, Fabiane SM, Sutton BJ, Calvert RA. The crystal structure of an avian IgY-Fc fragment reveals conservation with both mammalian IgG and IgE [J]. Biochemistry,2009, 48(3): 558-562.
15.韩水仲,齐宝珠,赵津子,等.卵黄抗体在医药领域中应用的新趋势[J].中国兽医科学,2010,40(10):1093-1097.
16. Sunwoo HH, Wang WW, Sim JS. Detection of Escherichia coli O157: H 7 using chicken immunoglobulin Y [J]. Immunol Lett, 2006,106(2): 191-193.
17. Vasconcellos FA, Coutinho ML, Dasilva EF, et al. Testing different antigen capture ELISA formats for detection of Leptospira spp in human blood serum [J]. Trans R Soc Trop Med Hyg, 2009, 10(16): 1-6.
18. Shimitzu M, Nagashima H, Hashimoto K, et al. Egg yolk antibody(IgY) stability in aqueous solution with high sugar concentrations [J]. Food sci,1994, 59(3): 763-772.
19. Chang HM, Ou-Yang RF, Chen YT, et al. Productivity and some properties of immunoglobulin specific against Streptococcus mutans serotype c in chicken egg yolk (IgY) [J]. J Agric Food Chem, 1999, 47(1): 61-66.
20. Lee KA, Chang SK, Lee YJ, et al. Acid stability of anti-Helicobacter pyroli IgY in aqueous polyol solution [J]. J Biochem Mol Biol, 2002, 35(5): 488-493.
21.陈阿琴,杨志刚,俞颂东.卵黄免疫球蛋白及其应用研究进展[J].中国兽药杂志,2005,39(3):28-31.
22.李春辉,埚立君,俞颂东.卵黄免疫球蛋白及其应用研究进展[J].中国生物制品学杂志,2005,25(1):45-46.
23.戴万明,聂琳. 1627名学龄前儿童IgY口腔喷剂防龋效果观察[J].浙江预防医学,2008,20(11):56-58.
24.王祝玲,王强.抗龋IgY牙膏对乳牙防龋效果的临床研究[J].牙体牙髓牙周病学杂志,2008,18(6):33-34.
25. Fujibayashi T,Nakamura M,Tominaga A, et al. Effects of IgY against Candida albicans and Candida spp. Adherence and Biofilm Formation [J]. Jpn. J. Infect Dis, 2009, 62(5): 337-342.
26.胡国柱,聂荣庆,张进,等.抗牙龈卟啉单胞菌IgY的制备及抑菌效果[J].实用口腔医学杂志,2010, 26(3): 337-340.
27. Okuda K, Kato T, Naito Y. Protective efficacy of active and passive immunizations against experimental infection with Bacteroids gingivalis in ligated hamsters [J]. J Pent Res, 1988, 67(5): 807-811.
28. Takahshi N, Eisenhuth G, Lee I, et al. Immunoglobulins in milk from cows immunized with oral strains of Actinomyces, Prevotella, Porphyromonas, and Fusobacterium [J]. J Dent Res, 1992, 71(8): 1509-1515.
29. Yokoyama K, Sugano N, Rahman AK, et al. Activity of anti-Porphyromonas gingivalis egg yolk antibody against gingipains in vitro [J]. Oral Microbiol Immunol, 2007, 22(5): 352-355.
30. Yokoyama K, Sugano N, Shimada T, et al. Effects of egg yolk antibody against porphyromonas gingivalis gingipains in periodontitis patients [J]. J Oral Sci, 2007, 49(3): 201-206.