IgY牙膏对远缘链球菌黏附定居的影响
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摘要
目的:研究IgY牙膏对远缘链球菌体外黏附及体内定居的影响。
方法:实验一:①分别在培养远缘链球菌的胰蛋白酶消化大豆液体培养基(TSB)中加入浓度分别为2.5mg/ml、1.25 mg/ml、0.625 mg/ml、0.3125 mg/ml的特异性抗变形链球菌IgY抗体和IgY牙膏溶液及对照牙膏(不含IgY抗体,其他成分与IgY牙膏相同)溶液,厌氧培养24h后,用分光光度计测定细菌生长黏附状况。②远缘链球菌在含有特异性IgY抗体和IgY牙膏溶液的TSB培养基中厌氧培养24h,离心得菌体,制作电镜切片,透射电镜下观察其形态学变化。
实验二:将24只Wistar大鼠随机分为3组,每组8只,雌雄各半。第1周广谱抑菌,第2周在大鼠牙面上接种S.sobrinus6715,2次/d,50ul/次。从第3周开始按分组用不同的溶液涂抹大鼠牙齿,连续3周。处死大鼠,拔取磨牙,取细菌标本,用MSB和BHI平板定量培养,结果以远缘链球菌占细菌总数的百分比来表示。
结果:实验一:①IgY牙膏组与对照牙膏组比较,黏附率有统计学差异(P<0.05); IgY牙膏与0.3125 mg/ml IgY溶液对远缘链球菌的黏附抑制率之间无统计学差异(P>0.05)。②经特异性IgY抗体处理的远缘链球菌,细胞壁不完整,细胞壁最外层荚膜溶解消失;经IgY牙膏处理的远缘链球菌,除上述变化外,部分菌细胞结构破坏,菌体溶解。
实验二:IgY牙膏组与IgY组之间的差异无统计学意义(P>0.05);IgY牙膏组与对照牙膏组之间的差异有统计学意义(P<0.05),即IgY牙膏能明显降低远缘链球菌在大鼠磨牙上的定居。
结论:IgY牙膏对远缘链球菌的体外黏附具有抑制作用,这种抑制作用是由于IgY牙膏使细菌的细胞壁破坏,导致其黏附作用丧失。IgY牙膏能减少大鼠磨牙菌斑中远缘链球菌的比例,即减少远缘链球菌在体内的定居。
Objective:To investigate in vitro adhesion and colonization of streptococcus sobrinus incubated with IgY toothpaste in vivo in rats.
Methods:
Experiment One:①S.sobrinus were cultured with different concentrations of IgY (2.5mg/ml, 1.25 mg/ml,0.625 mg/ml,0.3125 mg/ml), IgY toothpaste and control toothpaste respectively. After culture,the adhesion of S.sobrinus was observed by spectrophotometer.②Cultured streptococcus sobrinus treated with anticarious IgY and IgY toothpaste was observed by transmission electron microscope(TEM).
Experiment Two: 24 Wistar rats were randomly divided into 3 groups. All rats were fed with sterilized water containing Ampicilin 0.25㎎/ml at the first week and incubated with S.sobrinus 6715 twice a day at the second week.At the third week,different solution were smeared on the surface of the molars for three weeks. Bacterium samples were scraped in order to determine the numbers of S.sobrinus by MS plates and general bacterium by BHI plates.Then measured the proportion of S.sobrinus to gross colony .
Results:
Experiment One:①The adhesion rate had significantly difference between IgY toothpaste and control toothpaste(P<0.05),but no significantly difference between IgY toothpaste and egg yolk IgY(0.3125 mg/ml) (P>0.05).②The capsule of cell wall was dissolved in anticarious IgY group. In IgY toothpaste group some cells were dissolved,other than the above changes.
Experiment Two:The proportion of S.sobrinus to gross colony had significantly difference between IgY toothpaste and control toothpaste(P<0.05),but no significantly difference between IgY toothpaste and egg yolk IgY(0.3125 mg/ml) (P>0.05).
Conclusion: IgY toothpaste can inhibit in vitro adhesion of S.sobrinus. IgY toothpaste could lead to the loss of the cell capsule of streptococcus sobrinus and influences the adhesive effect.IgY toothpaste can inhibit in vivo colonization of S.sobrinus.
方法:实验一:①分别在培养远缘链球菌的胰蛋白酶消化大豆液体培养基(TSB)中加入浓度分别为2.5mg/ml、1.25 mg/ml、0.625 mg/ml、0.3125 mg/ml的特异性抗变形链球菌IgY抗体和IgY牙膏溶液及对照牙膏(不含IgY抗体,其他成分与IgY牙膏相同)溶液,厌氧培养24h后,用分光光度计测定细菌生长黏附状况。②远缘链球菌在含有特异性IgY抗体和IgY牙膏溶液的TSB培养基中厌氧培养24h,离心得菌体,制作电镜切片,透射电镜下观察其形态学变化。
实验二:将24只Wistar大鼠随机分为3组,每组8只,雌雄各半。第1周广谱抑菌,第2周在大鼠牙面上接种S.sobrinus6715,2次/d,50ul/次。从第3周开始按分组用不同的溶液涂抹大鼠牙齿,连续3周。处死大鼠,拔取磨牙,取细菌标本,用MSB和BHI平板定量培养,结果以远缘链球菌占细菌总数的百分比来表示。
结果:实验一:①IgY牙膏组与对照牙膏组比较,黏附率有统计学差异(P<0.05); IgY牙膏与0.3125 mg/ml IgY溶液对远缘链球菌的黏附抑制率之间无统计学差异(P>0.05)。②经特异性IgY抗体处理的远缘链球菌,细胞壁不完整,细胞壁最外层荚膜溶解消失;经IgY牙膏处理的远缘链球菌,除上述变化外,部分菌细胞结构破坏,菌体溶解。
实验二:IgY牙膏组与IgY组之间的差异无统计学意义(P>0.05);IgY牙膏组与对照牙膏组之间的差异有统计学意义(P<0.05),即IgY牙膏能明显降低远缘链球菌在大鼠磨牙上的定居。
结论:IgY牙膏对远缘链球菌的体外黏附具有抑制作用,这种抑制作用是由于IgY牙膏使细菌的细胞壁破坏,导致其黏附作用丧失。IgY牙膏能减少大鼠磨牙菌斑中远缘链球菌的比例,即减少远缘链球菌在体内的定居。
Objective:To investigate in vitro adhesion and colonization of streptococcus sobrinus incubated with IgY toothpaste in vivo in rats.
Methods:
Experiment One:①S.sobrinus were cultured with different concentrations of IgY (2.5mg/ml, 1.25 mg/ml,0.625 mg/ml,0.3125 mg/ml), IgY toothpaste and control toothpaste respectively. After culture,the adhesion of S.sobrinus was observed by spectrophotometer.②Cultured streptococcus sobrinus treated with anticarious IgY and IgY toothpaste was observed by transmission electron microscope(TEM).
Experiment Two: 24 Wistar rats were randomly divided into 3 groups. All rats were fed with sterilized water containing Ampicilin 0.25㎎/ml at the first week and incubated with S.sobrinus 6715 twice a day at the second week.At the third week,different solution were smeared on the surface of the molars for three weeks. Bacterium samples were scraped in order to determine the numbers of S.sobrinus by MS plates and general bacterium by BHI plates.Then measured the proportion of S.sobrinus to gross colony .
Results:
Experiment One:①The adhesion rate had significantly difference between IgY toothpaste and control toothpaste(P<0.05),but no significantly difference between IgY toothpaste and egg yolk IgY(0.3125 mg/ml) (P>0.05).②The capsule of cell wall was dissolved in anticarious IgY group. In IgY toothpaste group some cells were dissolved,other than the above changes.
Experiment Two:The proportion of S.sobrinus to gross colony had significantly difference between IgY toothpaste and control toothpaste(P<0.05),but no significantly difference between IgY toothpaste and egg yolk IgY(0.3125 mg/ml) (P>0.05).
Conclusion: IgY toothpaste can inhibit in vitro adhesion of S.sobrinus. IgY toothpaste could lead to the loss of the cell capsule of streptococcus sobrinus and influences the adhesive effect.IgY toothpaste can inhibit in vivo colonization of S.sobrinus.
引文
[1]张平.变形链球菌和远缘链球菌致龋毒力因素的差异.国外医学口腔医学分册,1997,24(1):24-26.
[2] Hirose H,Hirose K,Isogai E,et al. Caries Res,1993,27(4):292.
[3]史俊南,杨荣鉴,王捍国.龋病抗体研制动态和奥丽汀健齿露的研究.牙体牙髓牙周病学杂志,2001,11(4):269-273.
[4]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.牙体牙髓牙周病学杂志,2004,14(2):98-100.
[5] Hamada S,Horikoshi T,Minami T,et al. Oral passive immunization against dental caries in ratsby use of hen egg yolk antibodies specific for cell. Associated glucosy transferase of streptococcus mutans. Infect Immun,1991,59(11):4161-4167.
[6] Otake S, Nishihara Y,Makimura M,et al. rotection of rats against dental caries by passive immunization with hen egg yolk antibodies(IgY).J Dent Res,1991,70(3):162-166.
[7] Hatta HK,Tsuda M, Ozeki M,et .Passive immunization against dental plaque formation in humans:effect of a mouth rinse contaming egg yolk antibodies(IgY)specific to Streptococcus mutans[J]Caries Res,1997,31(4):268-274.
[8]黄文学,樊明文,边专等.特异性抗变形链球菌鸡蛋黄抗体IgY对变形链球菌生长及黏附的影响.口腔医学纵横杂志,2002,18(2):82-83.
[9]樊明文,江千舟,边专等.特异性抗变形链球菌鸡蛋黄抗体IgY对定菌鼠龋齿发生的影响.华西口腔医学杂志,2003,21(5):339-341.
[10]文冰,周汝俊,杨健等.变形链球菌鸡蛋黄抗体对变形链球菌合成葡聚糖影响的实验研究.上海口腔医学,2002,11(2):141-143.
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[13] Fan Mingwen,Jiang Qianzhou,Bian Zhuan,et al. Isolation of egg yolk IgY rom hens immunized with S.mutans B29-33 and S.sobrinus6715. J omp Stomatol, 2000,16(3):193-194.
[14] Ellwood DC. Adhesion of microorganisms to surfaces[M]. London:Academic Press,1979,138.
[15] De soet JJ,Van Loveren,Lammens AJ,et al. Difference in Cariogenicity between fresh isolates of streptococcus sobrinus and streptococcus mutans. Caries Res,1991,25:116-122.
[16] OHO T, Shimazaki Y,Mitoma M,et al.Bovine milk antibodies against cell surface protein antigen PAC-glucosyctransferases fusion protein suppress cell adhesion znd alter glucan synthesis of streptococcus mutans. J Nutr,1999,129(10),1836-1841.
[17]樊明文.关于免疫防龋.中华口腔医学杂志,2006,41(5):279-281.
[18]于维先,胡晓春,宋印章.葡糖基转移酶多克隆抗体对远缘链球菌定局的影响.吉林大学学报,2004,30(3):413-414.
[19] Van Raamsdonk M, de Soet JJ, Boch-Tijhof CJ, et al. Effect of antibodies on chemiluminescence and on killing of streptococcus sobrinus by polymrophnuclear leukocytes. Oral Microbiol Immunol,1996,11:254-258.
[20]刘天佳主编.口腔疾病的微生物学基础.北京:人民卫生出版社,1999,43.
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[3]樊明文,主编.口腔医学新进展[M] .武汉:湖北科学技术出版社,2000.
[4] Lehner T,Caldwell J,Smith R. Local passive immunization by monoclonal antibodies against streptococcal antigenⅠ/Ⅱin the prevention of dental caries[J]. Infect Immun,1985,50(3):796.
[5] Ma JK,Smith R, Lehner T. Use of monoclonal antibodies in local passive immunization to prevent colonization of human teeth by Streptococcus mutans [J]. Infect Immun,1987,55(5):1274
[6] Ma JK,Hunjan M,Smith R,et al. An investigation into the mechanism of protection by local passive immunization with monoclonal antibodies against Streptococcus mutans[J]. Infect Immun,1990,58(10):3407.
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[9] van Raamsdonk M,de Soet JJ,Bosch-Tijhof,et al. Effect of antibodies on chemiluminescence and on killing of Streptococcus sobrinus by polymorphonuclear leukocytes[J]. Oral Microbiol Immunol,1996,11(4):254.
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[28] Otake S, Nishihara Y,Makimura M,et al. rotection of rats against dental caries by passive immunization with hen egg yolk antibodies(IgY).J Dent Res,1991,70(3):162-166.
[29] Hatta HK,Tsuda M, Ozeki M,et .Passive immunization against dental plaque formation in humans:effect of a mouth rinse contaming egg yolk antibodies(IgY)specific to Streptococcus mutans[J]CariesRes,1997,31(4):268-274.
[2] Hirose H,Hirose K,Isogai E,et al. Caries Res,1993,27(4):292.
[3]史俊南,杨荣鉴,王捍国.龋病抗体研制动态和奥丽汀健齿露的研究.牙体牙髓牙周病学杂志,2001,11(4):269-273.
[4]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.牙体牙髓牙周病学杂志,2004,14(2):98-100.
[5] Hamada S,Horikoshi T,Minami T,et al. Oral passive immunization against dental caries in ratsby use of hen egg yolk antibodies specific for cell. Associated glucosy transferase of streptococcus mutans. Infect Immun,1991,59(11):4161-4167.
[6] Otake S, Nishihara Y,Makimura M,et al. rotection of rats against dental caries by passive immunization with hen egg yolk antibodies(IgY).J Dent Res,1991,70(3):162-166.
[7] Hatta HK,Tsuda M, Ozeki M,et .Passive immunization against dental plaque formation in humans:effect of a mouth rinse contaming egg yolk antibodies(IgY)specific to Streptococcus mutans[J]Caries Res,1997,31(4):268-274.
[8]黄文学,樊明文,边专等.特异性抗变形链球菌鸡蛋黄抗体IgY对变形链球菌生长及黏附的影响.口腔医学纵横杂志,2002,18(2):82-83.
[9]樊明文,江千舟,边专等.特异性抗变形链球菌鸡蛋黄抗体IgY对定菌鼠龋齿发生的影响.华西口腔医学杂志,2003,21(5):339-341.
[10]文冰,周汝俊,杨健等.变形链球菌鸡蛋黄抗体对变形链球菌合成葡聚糖影响的实验研究.上海口腔医学,2002,11(2):141-143.
[11] Smith DJ ,King WF,Godiska R,et al. Passive Transfer of Immunoglobulin Y Antibody to streptococcus mutans Glucan Binding protein B Confer protection against Experimental Dental caries. Infect Immun,2001,69(5):3135-3142.
[12] Wu C,Yang R,Zhou J,et al. Production and characterization of anovel chicken IgY antibody raised against C-terminal peptide from human thymidine kinade. Immunol Methods,2003,277(1-2):157.
[13] Fan Mingwen,Jiang Qianzhou,Bian Zhuan,et al. Isolation of egg yolk IgY rom hens immunized with S.mutans B29-33 and S.sobrinus6715. J omp Stomatol, 2000,16(3):193-194.
[14] Ellwood DC. Adhesion of microorganisms to surfaces[M]. London:Academic Press,1979,138.
[15] De soet JJ,Van Loveren,Lammens AJ,et al. Difference in Cariogenicity between fresh isolates of streptococcus sobrinus and streptococcus mutans. Caries Res,1991,25:116-122.
[16] OHO T, Shimazaki Y,Mitoma M,et al.Bovine milk antibodies against cell surface protein antigen PAC-glucosyctransferases fusion protein suppress cell adhesion znd alter glucan synthesis of streptococcus mutans. J Nutr,1999,129(10),1836-1841.
[17]樊明文.关于免疫防龋.中华口腔医学杂志,2006,41(5):279-281.
[18]于维先,胡晓春,宋印章.葡糖基转移酶多克隆抗体对远缘链球菌定局的影响.吉林大学学报,2004,30(3):413-414.
[19] Van Raamsdonk M, de Soet JJ, Boch-Tijhof CJ, et al. Effect of antibodies on chemiluminescence and on killing of streptococcus sobrinus by polymrophnuclear leukocytes. Oral Microbiol Immunol,1996,11:254-258.
[20]刘天佳主编.口腔疾病的微生物学基础.北京:人民卫生出版社,1999,43.
[1]张举之,主编.口腔内科学[M] .第3版.北京:人民卫生出版社,1999.
[2]刘天佳,主编.口腔疾病的微生物学基础[M] .北京人民卫生出版社,1999.
[3]樊明文,主编.口腔医学新进展[M] .武汉:湖北科学技术出版社,2000.
[4] Lehner T,Caldwell J,Smith R. Local passive immunization by monoclonal antibodies against streptococcal antigenⅠ/Ⅱin the prevention of dental caries[J]. Infect Immun,1985,50(3):796.
[5] Ma JK,Smith R, Lehner T. Use of monoclonal antibodies in local passive immunization to prevent colonization of human teeth by Streptococcus mutans [J]. Infect Immun,1987,55(5):1274
[6] Ma JK,Hunjan M,Smith R,et al. An investigation into the mechanism of protection by local passive immunization with monoclonal antibodies against Streptococcus mutans[J]. Infect Immun,1990,58(10):3407.
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