【作者】 陈玉；

【导师】 朱剑东；

【作者基本信息】 中国医科大学 ， 口腔临床医学， 2007， 硕士

【摘要】 目的龋病是细菌感染性疾病，变形链球菌（Streptococcus mutans，以下简称变链菌）是目前所公认的主要的致龋菌之一。白芨是传统的中草药，临床应用广泛，毒副作用较小，具有杀菌、消炎作用。虽然白芨在防龋中药中有所提及，但是相关的实验研究很少。本研究通过白芨水煎剂（Bletille striata Reichb.f decoction，BSD）对变链菌生长、产酸、粘附以及产胞外多糖的影响，初步探讨中药白芨成为防龋药物的可能性。方法1、白芨水煎剂和变链菌标准菌液的制备取白芨55g，干燥，粉碎，水提取前冷浸1小时，用蒸馏水1430ml（26倍加水量）煎煮2次，共煎煮4小时。无菌4层纱布过滤杂质，合并滤液浓缩至55ml，此为1g／ml的BSD，备用。变链菌标准菌株按说明书溶解，放入牛心脑浸液（BHI）固体培养基中，厌氧环境下复苏48小时。之后将菌种接种于胰蛋白酶大豆（TS）液体培养基中，增菌18小时。涂片检查为纯培养后，将菌液在4℃下3000 r·min^-1离心15分钟，收集细菌，用无菌生理盐水洗菌2次，并用其调整，使菌悬液在540nm处吸光度等于1.0，备用。2、白芨水煎剂对变链菌生长的影响1g／ml的白芨水煎剂和已溶化的BHI固体培养基采用倍比稀释法，使最终培养基含药液浓度分别为250.00mg／ml、125.00mg／ml、62.50 mg／ml、31.25mg／ml、15.62mg／ml、7.81mg／ml、3.90mg／ml、1.95mg／ml、0.98mg／ml。按CLSI／NCCLS推荐的琼脂稀释法测定实验菌株的MIC值。3、白芨水煎剂对变链菌产酸作用的影响根据BSD对变链菌MIC测定结果，选取MIC值以下4个浓度梯度，配制含1％蔗糖的TS液体培养基。采用酸度计调定初始PH值为7.4，接种等量的变链菌悬液。用PHS-25数显PH计测定培养物上清液的PH值，并求出培养前后的△PH值。实验共分5组（4个不同浓度的实验组和1个对照组），每组4个平行管，每管测定3次，结果取平均值。4、白芨水煎剂对变链菌粘附作用的影响采用比浊法。选取白芨水煎剂MIC值以下4个浓度，每管中插入已灭菌的直径为0.8mm牙用不锈钢丝一根及等量菌悬液，厌氧（80％N₂、10％CO₂、10％H₂）培养24小时后换上新鲜培养液，同等条件下再培养24小时；取出钢丝，将钢丝上粘附的细菌洗脱下来，分别测OD值。实验共分6组（4个不同浓度的实验组和阴性、阳性2个对照组），每组4个平行管，每管测定3次，结果取平均值。5、白芨水煎剂对变链菌产胞外多糖作用的影响本研究采用葸酮测多糖技术，测量BSD对变链菌产水溶性（样品A）、水不溶性（样品B）胞外多糖的影响。实验共分5组（4个不同浓度的实验组和1个对照组），每组4个平行管，每管测定3次，结果取平均值。6、统计学处理实验数据由SPSS 10.00软件进行统计学处理，结果以（?）±s表示，各组总体均数之间用单因素方差分析进行比较（Analysis of variance，ANOVA），各样本组间均数的比较采用SNK（Student-Newman-Keuls）。P＜0.05表示差异具有统计学意义。结果1、抑菌实验BSD浓度在62.50mg／ml以上的培养基内无变链菌生长，浓度在31.25mg／ml以下的培养基内均有变链菌生长，且随着浓度的降低，变链菌的菌株增多。根据实验结果判定其MIC值是62.50mg／ml。2、产酸实验随着BSD浓度的增加，实验前后△PH减少，说明变链菌产酸随浓度的增加而减少。经统计学处理，除3.90mg／ml浓度组外，各浓度组实验前后△PH值与对照组相比有显著性差异（P＜0.05）。3、粘附实验随着BSD浓度的降低，粘附抑制率明显减小。经统计学分析，除3.90mg／ml浓度组外，其它各浓度组吸光度值总体均数间及与对照组之间有统计学差异（P＜0.05）。4、产胞外多糖实验随着BSD浓度的增加，样品A、样品B的吸光度同时减少，而吸光度与胞外多糖的量呈正比关系，故随着浓度的增加，产水溶性、水不溶性胞外多糖的量也随着减少。其抑制作用随着浓度的增加而增强。经统计学分析，除3.90mg／ml、7.81mg／ml浓度组外，15.62mg／ml、31.25mg／ml浓度组与对照组之间有显著性差异P＜0.05。结论白芨水煎剂具有抑制变链菌生长、产酸、粘附和产胞外多糖的作用，有望成为新一代的中药防龋剂。更多还原

【Abstract】 ObjectiveDental caries is fundamentally a microbial disease which involves the destructionof hard tissue of tooth. Streptococcus mutans(S.mutans) is the main cariogenicbacteria. Bletille striata Reichb.f(BS) is a traditional Chinese medicine which isfrequently used in clinical practice, which was referred to caries-preventing traditionalChinese medicine. Research related to anti-caries was few. Aim of this study is to provethat BS may be employed as a new caries-preventing traditional Chinese medicine.Methods1．Bletille striata Reichb.f decoction(BSD) and S.mutans preparations55g BS (?) dried and crashed (?) soaked 1h(add 26 multiple amount cold water) (?)decocted twice, 4h (?) filterred with 4 layer sterilized sponge (?) 55ml BSD (1g/ml).S.mutans were dissolved according to instruction (?) cultivated in BHI 48h(37(?),80%N,10%CO,10%H) (?) cultivated in TS 18h(37(?),80%N,10%CO,10%H) (?)centrifuged 15min (?) washed twice with sterilized normal sodium (?) adapted theoptical density(OD) at 540nm to 1．0．2．Effect of BSD on growth of S.mutansUsing agar dilution test (?) BSD were doubly diluted to different concentrationsfrom 250．00mg/ml to 0．98mg/ml with melted BHI.3．Effect of BSD on acidogenicity of S.mutansSelected four different concentration BSD which were sub-MIC (?) added sucrose(?) 1% sucrose BSD (?) adapted every of them initial PH to 7．4 with PH meter (?) added identical bacteria (?) cultivated 48h(37(?), 80%N,10%CO,10%H) (?) measured PH ofsupernatants and calculated (?)PH.4．Effect of BSD on initial adhesion of S.mutansSelected four different concentration BSD which were sub-MIC (?) added identical0．8mm stainless steel wire and bacteria (?) cultivated 48h(37(?),80%N,10%CO,10%H) (?) washed bacteria on wire (?) measured OD.5．Effect of BSD on exopolysaccharides generation of S.mutansAnthrone method was used to study the effect of BSD on the content ofWSG(sampleA) and WIG(sampleB).6．Statistical analysisAll datum were analysed by SPSS 10．00 software.Comparison of total sample ofall groups was used by Analysis of variance (ANOVA). Student-Newman-Keulsmethod was used to compare the difference between each two groups. The significantlevel was set at 0．05．Results1．S.mutans did not appear in agar BHI which concentration is over 62．50mg/ml.S.mutans growed differently in agar BHI which concentration is sub 31．25mg/ml. Theywere concentration dependent.The lower concentration,the more S.mutans.MIC of BSDon S.mutans was 62．50mg/ml as determined by agar dilution test.2．(?)PH gradually decreased with the increasing concentration of BSD.Except forthe group of 3．90mg/ml,(?)PH of other groups were significantly different with that ofcontrol group(P<0．05) .3．Inhibitory rate significantly decreased with the concentration descend of BSD.Except for the group of 3．90mg/ml, OD of other groups were significantly differentwith that of control group and among the test groups (P<0．05) .4．With the concentration of BSD increasing,OD of sample A and B decreased atthe same time. So the WSG and WIG The more OD,the more exopolysaccharides generation. Except for the groups of 3．90mg/ml and 7．81mg/ml, 15．62mg/ml and31．25mg/ml were significantly different with control group (P<0．05) .ConclusionBletille striata Reichb.f decoction has inhibitory effect on the growth, adhesion,acidogenicity and exopolysaccharides generation of Streptococcus mutans. It may beemployed as a new caries-preventing traditional Chinese medicine.更多还原