远缘链球菌黏附与游离状态下合成胞外多糖的比较

目的：比较远缘链球菌在游离和黏附状态下合成水溶性、水不溶性多糖的能力并探讨其原因。方法：在不同培养时间和多种蔗糖浓度条件下，对远缘链球菌进行摇动培养和静止培养，以模拟该菌在游离和黏附的状态下生长。采用蒽酮法测定细菌合成的水溶性和水不溶性多糖的含量，实验数据采用SPSS10.0软件包分析，选用单因素方差分析的Dunnett双侧检验比较组间的差异有无统计学意义。结果：生物膜形成发展期，游离菌合成的水溶性多糖随培养时间的延长而增多，黏附菌合成的水溶性多糖则随时间呈下降趋势；在生物膜成熟期，黏附菌合成的水不溶性多糖多于游离菌（P〈0．05），而两者合成的水溶性多糖量的差异无显著性（P〉0．05）。细菌合成的水溶性、水不溶性多糖随蔗糖浓度的增加而增加；在各个蔗糖浓度条件下，黏附菌合成的水不溶性胞外多糖量显著多于游离菌（P〈0.05），而水溶性胞外多糖的量差异不大（P〉0．05）。结论：培养时间和蔗糖浓度增加，导致远缘链球菌合成的水不溶性胞外多糖较游离状态明显增加；生物膜特殊的生长环境，是造成这种差异的可能原因。     

不同蔗糖浓度下外源性右旋糖酐酶协同氟化钠对变异链球菌成熟生物膜和胞外多糖的作用研究

目的研究不同蔗糖浓度培养基下右旋糖酐酶(Dex)与氟化钠(NaF)联用对变异链球菌(S.mutans)成熟生物膜的消解作用和胞外多糖的调控作用。方法 (1)体外形成成熟生物膜,测量生物膜干重;(2)采用闪烁计数法检测不同蔗糖浓度下Dex和NaF对成熟生物膜胞外水溶性葡聚糖和水不溶性葡聚糖含量的影响;(3)扫描电子显微镜观察处理因素对成熟生物膜的消解作用。结果 (1)Dex+NaF作用后,生物膜干重与对照组相比显著降低(P<0.05);(2)Dex+NaF能显著减少S.mutans胞外多糖含量(P<0.05);(3)Dex+NaF对S.mutans成熟生物膜有明显消解作用(P<0.05);(4)Dex+NaF对胞外多糖和生物膜的消解作用随蔗糖浓度升高而增强。结论 Dex和NaF联用对S.mutans成熟生物膜具有消解作用,能显著减少其胞外多糖含量,作用效应随蔗糖浓度增加呈现增强的趋势。     

不同生存状态下变形链球菌合成胞外多糖的研究

目的 :比较变形链球菌合成水溶性、水不溶性胞外多糖的能力在生物膜黏附状态与浮游状态下是否存在量的差异及其可能原因。方法 :在多种蔗糖培养浓度和不同培养时间 ,对变形链球菌分别进行静置培养和摇动培养 ,以模拟生物膜黏附状态和浮游状态下生长的变形链球菌 ,蒽酮法测定细菌合成的水溶性胞外多糖和水不溶性胞外多糖的含量。结果 :相同培养时间每种蔗糖浓度条件下 ,生物膜黏附状态下变形链球菌合成的水溶性、水不溶性胞外多糖均要多于浮游状态下的细菌 (P <0 .0 5 ) ,生物膜黏附状态与浮游状态下变形链球菌的数量和合成胞外多糖的量均随着时间的增加而增加 ,不同培养时间同一蔗糖浓度条件下 ,生物膜状态下变形链球菌合成的水溶性、水不溶性胞外多糖也多于浮游状态下的细菌 (P <0 .0 5 )。结论 :生物膜黏附条件下 ,变形链球菌合成水溶性、水不溶性胞外多糖的能力均明显增强 ,这可能是生物膜中的细菌有更强致龋性的原因之一。     

高龋及无龋者变形链球菌临床分离株致龋性研究Ⅱ合成细胞外多糖能力的实验研究

目的：探讨来自不同龋敏感者的变形链球菌（血清型ｃ）临床分离株合成细胞外多糖的能力。方法：采用红外光谱分析对葡萄聚糖样本作定性分析,用蒽酮法分别定量测定水溶性和水不溶性的葡聚糖含量。结果：同一个体所带不同基因型变形链菌菌株合成细胞外多糖能力具有差异;高龋组个体定植的合成水溶性及水不溶性葡聚糖能力强的菌株所占比较显著高于无龋组。结论：高龋组变形链球菌（血清型ｃ）临床菌株的高致龋力与其携带有合成细胞外多糖能力强的菌株密切相关。     

高果糖玉米糖浆对变形链球菌合成细胞外多糖能力的影响

目的 应用蒽酮法,分别测定和比较变形链球菌代谢高果糖玉米糖浆（high-fructose cornsyrup,HFCS）和蔗糖所合成的细胞外多糖的含量,研究变形链球菌利用高果糖玉米糖浆合成细胞外多糖的能力。方法 配制质量浓度为0.25%、0.5%、1%、3%和5%的高果糖玉米糖和蔗糖培养基,将变形链球菌UA159接种其中,37℃微需氧培养24h后,用蒽酮法测定各培养基中所合成的细胞外水溶性和水不溶性多糖的含量。结果 除0.25%组和0.5%组中HFCS和蔗糖培养基中所合成的胞外水溶性多糖没有差异（P > 0.05）外,其它各组两培养基中所合成的胞外水溶性多糖均有差异,且蔗糖明显高于HFCS（P < 0.05）。5组浓度的HFCS组和蔗糖培养基中所合成的胞外水不溶性多糖均存在差异且蔗糖组明显高于HFCS（P < 0.05）。结论 在本实验设定的糖浓度范围内,变形链球菌利用高果糖玉米糖浆合成细胞外多糖的能力尤其是合成细胞外水不溶性多糖的能力较蔗糖弱。     

伊犁黑蜂蜂胶对口腔主要致龋细菌生物膜作用的实验研究

目的 研究伊犁黑蜂蜂胶对口腔常见致龋细菌生物膜的影响,探讨其防龋效果及可能的防龋机制。方法 通过结晶紫染色法测定黑蜂蜂胶对口腔常见致龋菌（变异链球菌、表兄链球菌、血链球菌、黏性放线菌、内氏放线菌）的最小生物膜清除浓度（MBEC）;培养测试细菌24 h单菌生物膜,加入MBEC及以下的3个浓度配置成初始pH值为7.0的含药培养基,厌氧培养24 h后测pH值,并计算pH变化值以检测不同浓度黑蜂蜂胶对测试菌单菌生物膜产酸能力的影响。蒽酮法测定MBEC及以下的3个浓度的黑蜂蜂胶对变异链球菌24 h单菌生物膜产生水不溶性胞外多糖的影响。结果 黑蜂蜂胶对变异链球菌、表兄链球菌、血链球菌、黏性放线菌、内氏放线菌的MBEC分别是6.25、1.56、3.13、0.78、0.78 mg•mL-1;黑蜂蜂胶可使各测试菌单菌生物膜ΔpH降低,蜂胶各组与对照组相比差异均有统计学意义（P<0.05）;在MBEC浓度时,蜂胶可使变异链球菌合成水不溶性胞外多糖的能力降低。结论 伊犁黑蜂蜂胶具有一定的防龋效果,其可能的防龋机制是通过有效清除口腔主要致龋细菌单菌生物膜,抑制测试菌株产酸、合成水不溶性胞外多糖的能力起作用的。     

Effect of Magnolia Officinalis Extract on Cariogenic Activity of Mutans Streptococci

目的：从中药厚朴中提取抗变链活性成分,并研究其对变形链球菌致龋特性的影响。方法：本研究应用薄层层析,萃取分离,硅胶色谱柱层析等现代中药化学实验方法,从生药厚朴中提取出有效成分MO2,用微量液体稀释法检测不同血清型变形链球菌对MO2的敏感性。以S.mutans MT703和S.sobrinus B13为实验菌株,分析MO2对其细胞表面疏水能力和合成水不溶性葡聚糖能力的影响。结果：MO2能降低细胞表面疏水率,抑制葡糖基转移酶催化合成水不溶性葡聚糖。随着MO2浓度的升高,细胞表面疏水率下降;80μg/ml的MO2对S.mutansMT703合成水不溶性葡聚糖的抑制率为45.4%,对S.sobrinusB13合成水不溶性葡聚糖的抑制率达43.5%。结论：厚朴提取物MO2对变形链球菌致龋能力有较强抑制作用。     

柠檬精油、柠檬烯、茶多酚对变形链球菌表面疏水性及黏附的影响

目的：研究天然植物成分柠檬精油（LEO）、柠檬烯（LIM）及茶多酚（TP）对变形链球菌（S．mutans）细菌表面疏水性及黏附能力的影响。方法：取最小抑菌浓度（MIC）以下浓度作为实验浓度;采用微生物黏着碳氢化合物法（MATH）,测定 S．mu-tans 表面疏水性;采用96孔板结晶紫染色法,评价 S．mutans 的黏附。结果：LEO、LIM、TP 在低于最小抑菌浓度（MIC）时,对S．mutans 表面疏水性及粘附具有抑制作用;在一定范围内其抑制作用随浓度增大而逐渐增加（P ＜0．05）;1／2 MIC 和1／20 MIC 时 LEO 抑制表面疏水性的作用强于 LIM和 TP（P ＜0．05）。结论：LEO 具有防龋应用前景。     

赤芍粗提物对变形链球菌影响的实验研究

目的研究赤芍粗提物对变形链球菌的生长、产酸、粘附及合成胞外多糖的影响。方法采用倍比稀释法测定不同浓度赤芍粗提物抑制变形链球菌生长的情况,通过统计学方法确定最低抑菌浓度（minimal inhibitory concentration,MIC）;再以MIC及低于MIC的4个倍比稀释浓度配制含药的胰酶解酪蛋白-植物蛋白胨-酵母提取物（trypticase-phytone-yeast extract medium,TPY）液体培养基,测定赤芍粗提物对变形链球菌产酸、粘附及合成胞外多糖能力的影响。结果赤芍粗提物抑制变形链球菌体外生长的MIC为12.5g/L;当赤芍粗提物浓度≥3.13g/L时有较明显地抑制变形链球菌产酸、粘附、合成水溶性胞外多糖的作用;当赤芍粗提物浓度≥6.25g/L时能明显抑制变形链球菌合成水不溶性胞外多糖。结论达到一定浓度的赤芍粗提物对变形链球菌的生长、产酸、粘附及合成胞外多糖均有一定的抑制作用。     

两种多孔生物玻璃支架材料的体外细胞相容性研究

目的 对比研究采用同样原料但以不同方法制备的两种多孔生物玻璃支架材料的体外细胞相容性。方法 溶胶－凝胶法制备A/W生物活性微晶玻璃（4006材料）,熔融法制备多孔生物活性玻璃（45S5材料）。体外诱导分离培养及鉴定兔骨髓基质细胞（BMSCs）,通过材料浸提液细胞毒性实验（MTT法）、细胞黏附实验、倒置相差显微镜、扫描电镜和环境扫描电镜比较4006材料和45S5材料对BMSCs细胞黏附和生长的影响,并探索与材料复合的最佳BMSCs细胞悬液浓度。结果 4006材料浸提液培养1 d时,其细胞活力与纯培养液间无统计学差异（P>0.05）;但培养3 d后,其细胞活力低于纯培养液（P<0.01）。45S5材料浸提液培养的细胞活力明显低于纯培养液（P<0.01）。细胞与材料复合培养后,镜下见BMSCs在4006材料孔隙内贴壁生长良好,分泌基质活跃;而在45S5材料上细胞黏附生长较差。BMSCs与4006材料的黏附量随接种细胞浓度升高而升高,细胞悬液浓度为2×10⁷个•mL^-1时的细胞黏附量最高。结论 溶胶－凝胶法制备的A/W生物活性微晶玻璃具有良好的生物活性和细胞相容性,具有作为骨组织工程支架材料的潜能。与其复合的细胞悬液浓度需要2×107 个•mL-1或以上。     

血型链球菌和变形链球菌葡糖基转移酶的对比分析

通过对血型链球菌（以下简称血链菌）３４和变形链球菌（以下简称变链菌）Ｉｎｇｂｒｉｔｔ（ｃ）葡糖基转移酶（ＧＴＦ）的活性测定，以及合成胞外多糖的对比分析，得出以下结论：（１）在相同培养条件下，提取血链菌３４的胞外ＧＴＦ的酶蛋白量比变链菌Ｉｎｇｂｒｉｔｔ（ｃ）多，而变链菌Ｉｎｇｂｒｉｔｔ（ｃ）胞外ＧＴＦ的酶活性高于血链菌３４；（２）血链菌３４的胞外ＧＴＦ酶具有合成ＩＧ和ＳＧ的能力，与变链菌Ｉｎｇｂｒｉｔｔ（ｃ）的胞外ＧＴＦ酶一样具有多形性，血链菌可以催化形成足够的葡聚糖来激活变链菌引物依赖型的ＧＴＦ酶，对牙菌斑的后继定殖菌是非常有利的；（３）获取血链菌３４的ＧＴＦ纯酶制剂比变链菌Ｉｎｇｂｒｉｔｔ（ｃ）容易。     

Study on the adhesion of an endemic strain of Streptococcus mutans serotype C to acquired pellicle. II. Isolation and extraction of adhesins of an endemic strain of Streptococcus mutans serotype C]

Surface proteins of S. mutans WD9463 A(c) were separated by DEAE-Sephadex A25 Chromatography and Sepharose C1-6B Chromatography. The adhesins were distinguished by bacterial adhesin inhibition experiment and were identified by PAGE, SDS-PAGE, IEP-PAGE, immunol diffusion test, GTF activity test and annalization of the sorts of amino acid. The results showed P1, two proteins with molecular weight of 117 kD and 127 kD and two proteins with GTF activity could inhibit the adhesion effectively. On the other hand, another kind of GTF could improve the adhesion of the strain. These indicated that S. mutans may be many kinds of adhesins.     

Contributions of three glycosyltransferases to sucrose-dependent adherence of Streptococcus mutans

Streptococcus mutans produces 3 types of glucosyltransferase (GTF), whose cooperative action is considered to be essential for its cellular adherence to the tooth surface. However, the precise mechanisms for synthesizing adhesive glucans and the specific roles of each GTF in cellular adherence to smooth surfaces have not been elucidated. In the present study, seven types of isogenic mutants of S. mutans MT8148 lacking GTFB, GTFC, and/or GTFD activities were constructed by inactivation of the genes encoding GTFB, GTFC, and/or GTFD. Furthermore, recombinant GTFB, GTFC, and GTFD were prepared from Escherichia coli cells harboring recombinant plasmids containing each of the gtf genes. Using these GTF-deficient mutants and rGTFs, we reconstituted sucrose-dependent adherence of S. mutans resting cells and examined the role of each GTF in vitro. The highest level of sucrose-dependent adherence was found at the ratio of 20 rGTFB:1 rGTFC:4 rGTFD in both the resting cells of GTF-deficient mutants and insoluble glucan synthesized by rGTFs. Moreover, when rGTFC and rGTFD were both present at concentrations of 1.5 mU and 6 mU, respectively, the insoluble glucan synthesized from sucrose by the rGTFs showed a high level of adhesiveness to smooth surfaces, even without rGTFB. These results suggest that the presence of all three GTFs at the optimum ratio is necessary for sucrose-dependent adherence of S. mutans, and that GTFC and GTFD may play significant roles in the synthesis of adhesive and insoluble glucan from sucrose.     

绿茶多酚和氟化钠对变链菌形态学及酶学的影响

目的：为深入探讨绿茶多酚防龋机理。方法：以氟化钠为对照,观察了绿茶多酚对变链菌形态学及其蔗糖酶、葡糖基转移酶（ＧＴＦ）和乳酸脱氢酶（ＬＤＨ）的影响,同时检测了培养液中蛋白含量和ｐＨ值的改变。结果：绿茶多酚和氟化钠对ＧＴＦ均有抑制作用,前者强于后者,而对蔗糖酶影响不明显。绿茶多酚对ＬＤＨ无明显影响,不能阻止培养基ｐＨ的下降,对菌液蛋白总量无明显改变;而氟化钠对ＬＤＨ有影响,明显减少培养基ｐＨ值的下降,且使菌液蛋白总量明显增加。结论：绿茶多酚对ＧＴＦ具有很强的抑制作用,这是其抗变链菌致病作用的主要途径之一。     

免疫乳清对葡萄糖基转移酶活性的影响

目的 了解免疫乳清对变形链球菌葡萄糖基转移酶（ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ,ＧＴＦ）活性的影响。方法 免疫乳清来源于ＧＴＦ过表达株Ｂ－２９－３３免疫孕牛中获得的牛奶。对照乳清从未免疫孕牛中获得。用ｇｔｆＢ基因缺陷株Ｂ－２９吸收免疫乳清中的抗体获得免疫吸收乳清。采用蒽酮定糖法检测３种乳清在５０、７０及９０μｌ３种剂量条件下对ＧＴＦ合成水不溶性葡聚糖的影响。结果 对照乳清具有增强酶活性的作用     

Streptococcus mutans glucosyltransferase: isolation and its ability to synthesize polysaccharides

The extracellular glucosyltransferases (GTF, EC 2.4.1.5) were extracted from trypticase yeast L-cystine culture supernatant of streptococcus mutans strains SSMC 167(g), 6715(g), SSMC 100 (c) and Ingbritt (c) by 60% saturated ammonium sulphate precipitation. The yield was more than 50%. All the enzyme preparations could synthesize both water-soluble and insoluble glucans. The results of polyacrylamide gel electrophoresis of enzyme preparations illustrated the complexity and multicomponent nature of the GTF of S. mutans. The abilities of synthesizing polysaccharides were different between S. mutans serotype g and serotype c. The ability of synthesizing water-soluble glucan was almost the same as that of synthesizing water-insoluble glucan for GTFs from the two S. mutans strains of serotype g, but the ability of synthesizing water-soluble glucan was 10-fold to that of synthesizing water-insoluble glucan for GTFs from other two strains serotype c. No frucotosyltransferase activity was found.     

葡糖基转移酶合成肽疫苗的免疫原性研究

目的　检测合成的葡糖基转移酶 (glucosyltransferase ,GTF)多肽疫苗的免疫原性 ,有助于研制以合成多肽为基础的防龋疫苗。方法　合成融有GTF催化和葡聚糖结合区的 2 7个氨基酸残基肽段 ,利用ELISA法检测免疫小鼠抗体的产生 ,通过GTF酶活性与变形链球菌粘附实验测定其抗血清的作用。结果　融合多肽疫苗的序列为ANDVDNSNPVVQAEQLYFRANGVQVKG ,免疫小鼠脾脏重量显著增加 ,可有效诱导机体抗体的产生。其免疫血清不仅拮抗GTF的酶活性 ,而且明显抑制变形链球菌的粘附。结论　GTF多肽疫苗可产生抗体介导的抑制GTF酶活性和抑制葡聚糖结合作用 ,对龋病的防治十分有价值     

Influence of cranberry juice on glucan-mediated processes involved in Streptococcus mutans biofilm development

Cranberry juice (CJ) has biological properties that may provide health benefits. In this study, we investigated the influence of CJ (pH 5.5) on several activities in vitro associated with the development of Streptococcus mutans UA159 biofilms. The ability of CJ to influence the adherence of S. mutans to either saliva- (sHA) or glucan-coated hydroxyapatite (gsHA), and to inhibit the glucan production by purified glucosyltransferases adsorbed to sHA was determined. For the adherence assays, we used both uncoated and saliva-coated bacterial cells. Furthermore, we examined whether CJ interferes with the viability, development, polysaccharide composition and acidogenicity of S. mutans biofilms. A solution containing equivalent amounts of glucose, fructose and organic acids at pH 5.5 was used as negative control. The adherence of S. mutans (uncoated and saliva-coated) to either sHA or gsHA treated with 25% CJ (v/v) was remarkably reduced (40-85% inhibition compared to control: p < 0.05), indicating that CJ effectively blocked the bacterial adherence to binding sites in salivary pellicle and in glucans. In contrast, when the bacterial cells alone were treated with CJ they adhered to the similar untreated surfaces. Cranberry juice (25%, v/v) also inhibited the activities of surface-adsorbed GTF B and C (70-80% inhibition compared to control, p < 0.05). The effect of CJ on the viability of microorganisms in biofilms was not significant. Biofilm formation and accumulation were significantly reduced by topical applications of 25% CJ (v/v) twice daily with 1-min exposures (p < 0.05). The biomass and insoluble glucan content of the biofilms in addition to its acidogenicity were significantly reduced by cranberry treatments (p < 0.05). Our data show that cranberry juice inhibited glucan-mediated biofilm development and acid production, and holds promise as a natural product to prevent biofilm-related oral diseases.     

酸性环境对变形链球菌黏附力的影响

目的 研究酸性环境对变形链球菌黏附能力的影响.方法 课题组前期获得的变形链球菌(血清C型)临床分离株20株及参考株UA159.用唾液包被的羟磷灰石(saliva coated hydroxyapatite,SHA)模拟口腔中牙面情况.各菌株分别在含3H并且初始pH分别为5.5和7.0的1%葡萄糖TPY液体培养基中培养,然后配成细菌悬液.菌悬液与SHA作用90 min,清洗、吸干,液体闪烁计数法检测各样本中黏附于SHA的细菌量.黏附量以每分钟闪烁计数值(CPM)表示.结果 pH 5.5与pH 7.0组变形链球菌对SHA的黏附量分别为469.433 3±272.640 8和3 447.343±2 837.814 9,P＜0.05.结论 酸性环境抑制变形链球菌的黏附力.     

Inhibition of Streptococcus mutans adhesion to buccal epithelial cells by an aqueous extract of Thymus vulgaris

OBJECTIVES: The aim of this study was to investigate the effect of an extract of Thymus vulgaris (thyme) on the growth of Streptococcus mutans (S. mutans) and the adhesion of this bacterium to human buccal epithelial cells. METHODS: Different concentrations of an aqueous extract of thyme were prepared and the effects investigated on growth of S. mutans. Furthermore, the effect of these extracts on adhesion of S. mutans to buccal epithelial cells was also investigated and compared with the effects of chlorhexidine digluconate. RESULTS: The data revealed that exposure of S. mutans to thyme extract showed a time and concentration-dependent decrease in bacterial viability. The greatest effect was observed when S. mutans had been exposed to 20% thyme extract for a period of 48 h which resulted in 96% inhibition of bacterial growth. Furthermore, the adhesion of S. mutans to buccal epithelial cells was also reduced when either buccal epithelial cells or S. mutans had been pre-incubated with different concentrations of aqueous thyme extracts (83-98% and 75-89% inhibition respectively). There was also greater reduction in the adherence of bacterial cells to buccal epithelial cells after mouth rinsing with 20% aqueous thyme extract compared to rinsing with chlorhexidine digluconate (45% and 89% inhibition of bacterial adhesion respectively). CONCLUSION: The diminished adherence of S. mutans to buccal epithelial cells after exposure to various concentrations of aqueous thyme extract as well as the antimicrobial properties of this plant may have clinical relevance.