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金银花醇提液对根管内粪肠球菌作用的体外研究
【作者】 孙剑；
【导师】 许颖；
【作者基本信息】 佳木斯大学 ， 口腔临床医学， 2010， 硕士
【摘要】 目的:目前国内外研究都有报道粪肠球菌在牙髓病和根尖病中的致病性,本实验通过对比金银花与次氯酸钠对粪肠球菌的作用来验证其对粪肠球菌的抗菌能力。并进一步研究金银花醇提液对抗粪肠球菌生物膜及阻断粪肠球菌生物膜早期粘附的能力,为今后利用金银花有效成分对抗粪肠球菌感染提供理论基础。方法:分别采用平皿打洞法和直接接触法验证粪肠球菌对金银花醇提液的敏感性,直接接触实验中利用噻唑兰(MTT)辅助判断金银花醇提液的最小抑菌浓度(MIC),并通过涂皿培养验证该方法的准确性;建立粪肠球菌生物膜体外模型,通过金银花醇提液作用后,应用MTT比色法计算金银花醇提液对粪肠球菌生物膜的抑菌率。以碘化丙啶(PI)荧光标记死菌细胞核,在激光共聚焦显微镜(CLSM)下观察药物对生物膜中粪肠球菌的影响;将建模引导材料预先用200mg/ml浓度的金银花醇提液处理后进行建模,使用MTT检测样本的代谢活性并在CLSM下观察4小时后粪肠球菌粘附载体情况。结果:平皿打洞实验中800mg/ml金银花醇提液抑菌环直径最大为(22.60±0.95)与5.25%次氯酸钠(20.70±1.11)无显著统计学差异(P>0.05),接触试结果金银花醇提液MIC为200mg/ml。涂皿培养后MIC结果与使用MTT辅组法所得相同。金银花醇提液对粪肠球菌生物膜的抑菌作用随实验组药物浓度增加而增加,800mg/ml金银花醇提液组抑制率最高(81.16%),25mg/ml金银花醇提液组抑菌率最小(41.76%)。各实验组与阴性对照组相比均有显著统计学差异(P<0.05)。使用CLSM观察可见金银花醇提液可使粪肠球菌生物膜内活菌比例显著下降。金银花醇提液对粪肠球菌生物膜表层细菌作用效果明显,但对内层细菌即使较高浓度金银花醇提液作用后也有细菌存活。引导材料经药物处理后降低了粪肠球菌的粘附,MTT结果显示在代谢活性上实验组与对照组之间差异明显(P<0.05),CLSM下显示载体上粘附的粪肠球菌密度明显降低。结论:1金银花醇提液对粪肠球菌有明显抑制作用。2金银花醇提液对粪肠球菌生物膜具有一定抑制作用,对生物膜表层细菌效果显著,但即使使用较高浓度的金银花醇提液也不能完全杀死生物膜内层的细菌。3金银花醇提液可干扰粪肠球菌的粘附,有预防粪肠球菌生物膜形成的作用。
【Abstract】 Object:At present,research of domestic and foreign reported that the pathogenicity of E. faecalis in pulposis and periapical disease. In this study,by compared ethanol extracts of honeysuckle with sodium hypochlorite solution on Inhibition in E.faecalis to Verify it’s antimicrobial activity in E.faecalis .And further study ethanol extracts of honeysuckle against E.faecalis biofilm and and adhesion ability of E.faecalis In the early,theoretical basis for use of honeysuckle active ingredients against E.faecalis infection in the future.Method: plate-hole diffusion assay and direct contact test was used to verify E.faecalis on the sensitivity of ethanol extract of honeysuckle, the direct contact test was determined that the minimal inhibitory concentration(MIC) of ethanol extracts of honeysuckle on E.faecalis invitro using MTT assay assisted,verify the accuracy of this method with plate cultivation;Established E.faecalis mono-biofilm model,in vitro. After processed with ethanol extracts of honeysuckle,reckon the Inhibition rate of ethanol extracts of honeysuckle to E.faecalis mono-biofilm ,MTT assay was used.Fluorescent stained with propidium iodide(PI)and examined through used of CLSM,observed the effect of drugs on the E.faecalis mono-biofilm.adherent materials of biofilm pre-processed with 200mg/ml ethanol extracts of honeysuckle then established E.faecalis mono-biofilm model,Samples to be tested with metabolic activity using MTT assay,and examined through use of CLSM to observed the adhesion of E.faecalis on the adherent materials.Result:The 800mg/ml ethanol extracts of honeysuckle (22.60±0.95)was the most effective in the plate-hole diffusion assay ,it had no significant difference with 5.25% sodium hypochlorite solution(20.70±1.11)(P>0.05),The results of MIC for ethanol extracts of honeysuckle was 200mg/ml in direct contact test. The MIC result determined by plate cultivation was similar to the result by MTT assay assisted.Inhibitory effect of ethanol extracts of honeysuckle to E.faecalis mono-biofilm increased with the drug concentration.800mg/ml ethanol extracts of honeysuckle acquired maximum inhibition rate(81.16%),and 25mg/ml ethanol extracts of honeysuckle acquired minimum inhibition rate(41.76%).The results showed significant statistical difference (P<0.05)between the experimental group and the negative control group.The results showed ethanol extracts of honeysuckle could decrease the vitality of E faecalis in the biofilm significantly.The effect of ethanol extracts of honeysuckle was obvious to the bacterial in E.faecalis mono-biofilm surface,inner bacterial couldn’t be completely inactivated even higher concentrations ethanol extracts of honeysuckle was used.Adhesion ability of E.faecalis was significantly inhibited after adherent materials be pre-processed.The MTT result showed significant statistical difference (P<0.05) to metabolic activity between the experimental group and the control group.Conclusion:1. The ethanol extracts of honeysuckle showed satisfactory antibacterial activity against E.faecalis.2. The effect of ethanol extracts of honeysuckle was obvious to Inhibited E.faecalis mono-biofilm,effect especially to the bacterial in E.faecalis mono-biofilm surface,but Even with a higher concentration of physic liquor couldn’t completely killed the bacteria in biofilms.3. Ethanol extracts of honeysuckle could reduce the adhesion ability of E. faecalis. The results indicates that it could prevent the formation of E.faecalis mono-biofilm.