【作者】 蒋琳；

【导师】 林居红；

【作者基本信息】 重庆医科大学 ， 口腔临床医学， 2008， 硕士

【摘要】 背景:牙周炎是牙周组织炎症性疾病;菌斑细菌及其产物是牙周炎最主要的病因,是引发牙周炎必不可少的始动因子。成纤维细胞是牙周组织中最多、功能上最重要的细胞,任何原因所造成的成纤维细胞的破坏,均能影响牙支持组织的健康。故一种理想的牙周炎治疗药物,既要达到良好的抑菌效果,又要保护牙周组织,具有较小的毒性作用。目前第3代硝基咪唑类衍生物奥硝唑因其具有较强的抗厌氧菌活性,成为治疗牙周炎的主要抗菌药物,而抗生素的长期用药具有逐渐产生耐药性的潜在隐患。牙周炎作为一种多细菌混合感染、局部全身因素共同影响的疾病,一种抗厌氧菌的单一药物无法达到良好的治疗效果,现在普遍推崇联合用药方案。寻求一种可行且有效的联合用药方案成为牙周炎治疗领域的研究热点。近年来,蜂胶因其具有广泛的生物学活性和药理作用得到人们的关注。以往研究已证实蜂胶和部分抗生素具有抑菌协同作用,然而主要集中在蜂胶与大环内酯类药物、多肽类抗生素、氨基苷类抗生素、四环素和氯霉素类抗生素的联合用药研究,且对需氧菌,如金黄色葡萄球菌的抑菌活性研究较多。在牙周炎治疗领域,将蜂胶与牙周抗菌药物联合应用,研究其对牙周厌氧菌的抗菌活性,目前未见报道。考虑蜂胶广泛的药物学作用及协同抑菌的特点,将蜂胶与抗生素联合应用治疗牙周炎,有望成为一种新型配伍方案。目的:本研究在参考国内外研究的基础上,将国产水溶性蜂胶和奥硝唑联合,研究其对牙龈卟啉单胞菌(Pg)的体外生长抑制作用及对人牙龈成纤维细胞(HGF)的毒性作用,初步探索蜂胶与奥硝唑联合治疗牙周炎的可行性,为其更加深入地研究提供初步依据。材料与方法:1.蜂胶抑制Pg体外生长的实验研究。用心脑浸液(BHI)+Hemin培养基按2倍稀释法将蜂胶和奥硝唑分别稀释成8个浓度和14个浓度的药液。每个试管加入0.5ml标化的菌液(106～107CFU/mL),充分混匀后,厌氧培养48 h。观察试管的混浊情况,选择适当浓度段划线接种于BHI血琼脂平板上。经厌氧培养48 h,无细菌生长的最低药物浓度为最小杀菌浓度(MBC),设立阴性对照组、阳性对照组。重复实验3次。2.蜂胶奥硝唑联合抑制Pg体外生长的实验研究。采用纸片扩散法,将奥硝唑(0.5、0.4、0.3、0.2、0.1 g/L五个浓度)和蜂胶(100、50、25 g/L三个浓度)单独药敏片及各浓度结合后的合剂药敏片贴在接种了Pg的BHI血琼脂培养基平皿内。37℃厌氧培养48 h,测定每一药敏片的抑菌环直径。每种药敏片重复4次。3.蜂胶奥硝唑联合对HGF的细胞毒性研究。消化第5代细胞,接种于96孔板,设阴性对照组,在37℃,95%湿度和5%CO2的条件下培养24 h,再用含有各浓度蜂胶、蜂胶奥硝唑合剂或奥硝唑的培养液继续培养24 h,用MTT比色法测定吸光度值(A值)。计算出细胞相对增殖率(RGR)。每种试剂选择RGR约为50%的浓度组进行生长曲线的研究。消化第6代细胞,接种于96孔板,每15孔为一组,一共8组,培养24 h。换上含有试剂的培养液,继续培养24 h,选择第1组用MTT比色法测定A值。其余7组弃去含有试剂的培养液,换上新鲜培养液继续培养,连续7 d,每天选择1组进行A值测定。最后绘制各生长曲线,与阴性对照组曲线比较。结果:1.蜂胶水溶液对Pg具有较好的生长抑制作用,当浓度为1.56 g/L时,就无细菌生长,其作用与0.125～0.25mg/L的奥硝唑相当。2.100 g/L蜂胶的平均抑菌环大小仅为10.25mm;蜂胶与奥硝唑联合后,含100 g/L蜂胶的合剂1组的抑菌环大小均大于对应浓度的单独奥硝唑组,含50 g/L蜂胶的合剂2组和含25 g/L蜂胶的合剂3组在奥硝唑低浓度时的抑菌环大小也大于对应浓度的单独奥硝唑组。随着蜂胶浓度的增加,抑菌环增大。析因分析结果表明,蜂胶与奥硝唑对Pg的生长抑制作用存在协同作用。3.随着药物浓度的降低,HGF的RGR趋于100%,毒性级别趋于0。奥硝唑浓度为0.4g/L或0.8g/L时,合剂组的A值均高于对应浓度的奥硝唑组,差别有统计学意义(P<0.05);奥硝唑浓度为0.2g/L或1.2g/L时,三组比较差别无统计学意义(P>0.05)。4.1.2g/L奥硝唑组、1.2g/L奥硝唑和1g/L蜂胶合剂组、1.2g/L奥硝唑和0.5g/L蜂胶合剂组及4g/L蜂胶组的RGR均约为50%。换上不含药物的培养液继续培养,连续7 d测定每组A值,组内不同时间点比较,有统计学差别(P<0.05);各时间点内,实验组与阴性对照组比较,均有统计学差别(P<0.05),但随着时间推移,各实验组的A值均逐渐趋向于阴性对照组。结论:1.蜂胶对Pg具有良好的生长抑制作用,MBC为1.56 g/L。2.蜂胶奥硝唑联合对Pg的生长抑制作用优于奥硝唑及蜂胶的单独用药,表明蜂胶与奥硝唑对Pg的生长抑制作用具有协同作用。3. 1g/L和0.5g/L低浓度蜂胶不会增加奥硝唑对HGF的细胞毒性作用,甚至在奥硝唑部分浓度段,表现出降低奥硝唑细胞毒性作用的效果。4.HGF分别在含有1.2 g/L奥硝唑、1.2g/L奥硝唑和1g/L蜂胶合剂、1.2g/L奥硝唑和0.5g/L蜂胶合剂或4g/L蜂胶的培养液内培养24 h后,约50%的细胞存活,连续7 d,各组细胞数均持续增加,表明药物的细胞毒性作用可逆。5.蜂胶奥硝唑联合应用治疗牙周炎具有深入研究价值。更多还原

【Abstract】 Background:Periodontitis is an infection of the tissues around the teeth. Dental plaque is the key pathogenic factor of periodontitis.Fibroblast is the main cell in the periodontal tissues, showing the most important function. Any destroy to fibroblast will affect health of periodontal tissue. So an ideal medicine for periodontitis not only can kill or inhibit the growth of bacteria, but also is safe for periodontal tissue, lower toxicity.At present , nitromidazole medicine, like ornidazole is main antibacterial which shows ideal anti-anaerobic effect. The emergence of antibiotic resistance in pathogenic bacteria will happen with long-term use of antibiotic.And periodontitis is caused by multi-bacterium, so one anti-anaerobic medicine alone can not show the ideal therapeutic efficacy. So the combinaion of antibacterials for periodontitis is advocated highly.Recently, special attention has been paid to natural propolis, which . has versatile pharmacological activities. And some studies have found that propolis shows antimicrobial synergistic action with some antibiotics, such as macrolides, polypeptide antibiotics, aminoglycosides, ambramycin and amphemycin. These studies are mainly regarded aerobic bacteria.Now there is no study about propolis combined with periodontal antibacterial for anaerobic. Combining propolis and ornidazole possess value of being studied in treatment of periodontitis, which may a new type of compatibility program.Objective:Based on some domestic and abroad studies, we combine advantage of propolis and ornizaloe and explore potentiality of mixture combined them for treating periodontal disease. The synergism of propolis and ornidazole on porphyromonas gingival (Pg) and toxic action on human gingival fibroblast(HGF)will be evaluated, so that we can get experimental information of composition of propolis and ornidazole treating periodontitis.Material & Methods:1.Experiment of propolis to inhibit growth of Pg in vitro. With brain-heart infusion (BHI) medium added hemin, propolis was diluted to follow 8 concentrations by fluit delution method and ornidazole was diluted to 14 concentrations. 0.5 ml of bacteria suspension (106～107CFU/mL) was added into every test tube added above test agents respectively. After thoroughly mixtured, Pg was incubated anaerobicly for 48 hours, then streakly inoculated on BHI blood agar plates. After 48 hours, the minimal bactericidal concentration (MBC) of propolis and ornidazole were determined. The experiment was repeated three times and positive control group and negative control group were set up every time.2.Experiment of composition of propolis and ornidazole to inhibit growth of Pg in vitro. By the disc diffusion methods, paper discs respectively containing ornidazole (0.5, 0.4, 0.3, 0.2 or 0.1g/L) , propolis (100, 50 or 25g/L) ,or composition (containing propolis and ornidazole ) were pasted on BHI blood agar plates inoculating Pg. The plates were incubated anaerobicly at 35℃for 48 h. The diameters of the inhibitory zones were measured. Every medicine paper discs were repeated 4 times.3.Cytotoxicity of composition of propolis and ornidazole on HGF. The fifth generation cells were grown in 96-well for 24 h in condition of 37°C, 95% humidity, and 5% CO2, then culture medium in every well was removed, culture medium respectively containing propolis or ornidazole or composition was added into wells, and 96-well was cultured again for 24 h. Absorbance (A) value of each well was determined by MTT colorimetric method and relative growth rate (RGR) of HGF was calculated. We chose the concentration groups of every agents , RGR of which was all about 50%, then carried out experiment of determining the growth curve of Pg.. The sixth generation cells were grown in 96-well for 24 h (all 8 groups with each group 15 wells, each agent 3 wells). Then cells were cultured in culture medium containing required concentration of test agent for 24 h, A value of each well of the first group was determined by MTT colorimetric method. The wells of other 7 groups were added with fresh culture medium without test agents after the medium with test agent was removed from all wells. One group was selected out every day in the 7 days and A value was determined. Then the growth curves of Pg of every test agent group and negative control group were drew.Results:1.Propolis showed the good inhibiting effect on growth of Pg, with MBC value is 1.56 g/L. And MBC value of ornidazole to Pg was ranging from 0.125mg/L to 0.25mg/L.2.The mean of inhibiting zone of propolis group at concentration of 100g/L was just 10.25 mm. While after ornidazole combined with propolis, the inhibiting zones of composition groups with 100g/L propolis were all greater than those of ornidazole groups when comparing between the same concentration of ornidazole groups. This results also were showen in composition groups combined 50 g/L or 25 g/L propolis with low concentration of ornidazole. The inhibiting zones became big with increasing of concentration in each group. The statistic analytic result showed synergistic effect existed between propolis and ornidazole for inhibiting growth of Pg.3.The relative growth rate (RGR) of HGF was up to 100% and the class of toxicity was low to zero with decreasing of agent concentration. When concentration of ornidazole was 0.4g/L or 0.8g/L, the average A values of composite agent groups all exceeded those of oenidazole groups alone, and there were all statistical differences ( P < 0.05 ) .When concentration of ornidazole was 0.2g/L or 1.2g/L, there were no statistical differences between composite agent groups and ornidazole groups(P>0.05).4.RGRs of HGF all were about 50%,after 24-hour culture in the culture medium containing 1.2g/L ornidazole ,composite agent 1 (containing 1.2% ornidazole and 1g/L propolis ), composite agent 2 (containing 1.2% ornidazole and 0.5g/L propolis ) or 4g/L propolis. When cells continued to be cultured in the culture medium without agents, the A values of above four groups and negative control group would increased as time gone. There were statistical differences between time points of examination for the same group(P<0.05).At each point in time, there were statistical differences between each agent group and negative control group(P<0.05),but the A values of above four agent groups all closed to that of negative control group with passage of time.Conclusion:1.Propolis can inhibit the growth of Pg.2.The composition of propolis and ornidazole shows better growth inhibiting effect for Pg than ornidazole alone or propolis alone. Propolis and ornidazole can synergistically enhance growth inhibiting effect on Pg.3. 1g/L and 0.5g/L propolis can not enhance cytotoxic effect of ornidazole for HGF and can weak cytotoxic effect of ornidazole at some concentrations of ornidazole.4.About half of HGF could normally grow after cultured in the culture medium containing 1.2g/L ornidazole ,composite agent 1 (containing 1.2% ornidazole and 1g/L propolis ), composite agent 2 (containing 1.2% ornidazole and 0.5g/L propolis ) or 4g/L propolis for 24 hours. And the cell number in every group kept on increasing in 7 days, which indicated the cytotoxic effect of above agents for HGF is reversible.5.The composition of propolis and ornidazole is worth of be further studied in the respect of therapy of periodontitis.更多还原