目的 探索pH响应性新型叔胺单体(DMAEM)改性树脂粘接剂(DMAEM@RA)防治继发龋的应用前景.方法 按粘接剂改性的方法,向树脂粘接剂中添加5％DMAEM,改性合成DMAEM@RA.以变异链球菌(Sm)和干酪乳杆菌(Lc)双菌种生物膜为研究模型,分别在树脂粘接剂(对照组)和DMAEM@RA(DMAEM@RA组)上进行培养.分别设置pH值为7.4、6.0、5.5和5.0的培养体系.通过定量PCR分析菌量,通过扫描电镜和胞外多糖染色分析DMAEM@RA对双菌种生物膜表型、菌量及胞外多糖的影响.实时荧光定量PCR研究DMAEM@RA对Sm致龋毒力基因的影响.结果DMAEM@RA在酸性条件下可显著降低Sm和Lc的菌量,特别是Lc.pH=5.0时,DMAEM@RA组共培养 Sm 菌量对数值[lg(CFU/ml)](7.58±0.01)显 著低于对照组(7.87±0.03)(t=14.32,P＜0.001),Lc菌量对数值[lg(CFU/ml)](7.29±0.04)亦显著低于对照组(7.93±0.15)(t=6.93,P=0.002).扫描电镜下也可观察到DMAEM@RA组菌量减少,同时有细胞碎片出现.此外,DMAEM@RA可在酸性条件下显著减少双菌种生物膜的胞外多糖生物量,pH=5.0时DMAEM@RA组胞外多糖生物量值[(25.13± 3.14)mm3/mm2]显著低于对照组[(42.66±7.46)mm3/mm2](t=3.75,P=0.020).DMAEM@RA可在酸性条件下显著上调Sm的gtfB、gtfC基因表达,pH=5.0时gtfB、gtfC基因分别显著上调(14.64±0.44)和(2.99±0.20)倍(t=-42.74,P＜0.001;t=-13.55,P＜0.001).结论 DMAEM@RA在酸性条件下有较好的抑菌作用,具有防治继发龋发生发展的良好潜力.
Effect of a novel pH-responsive tertiary amine monomer dodecylmethylaminoethyl methacrylate modified resin adhesive on biofilm formation of Streptococcus mutans and Lactobacillus casei in vitro
Objective To explore the application prospect of a new pH-responsive tertiary amine monomer dodecylmethylaminoethyl methacrylate(DMAEM)modified resin adhesive(DMAEM@RA)in the prevention and treatment of secondary caries.Methods Five percents DMAEM was added to the resin adhesive to synthesize DMAEM@RA for modifying.Streptococcus mutans(Sm)and Lactobacillus casei(Lc)biofilms were cultured on resin adhesive and DMAEM@RA,respectively.The culture systems were set up at pH=7.4,6.0,5.5,and 5.0.The antimicrobial activity of DMAEM@RA was evaluated by quantitative PCR.The effects of DMAEM@RA on biofilm thickness,bacterial amount,and extracellular polysaccharides were studied by scanning electron microscope(SEM)and extracellular polysaccharide staining.Real-time fluorescence quantitative PCR was used to study the effect of DMAEM@RA on the expression levels of cariogenic genes in Sm.Results DMAEM@RA could significantly reduce the amount of Sm and Lc under acidic conditions,especially Lc.At pH=5.0,the logarithm value of co-cultured Sm bacteria[lg(CFU/ml)]in DMAEM@RA group(7.58±0.01)was significantly lower than that in control group(7.87±0.03)(t=14.32,P＜0.001),and the logarithm value of Lc bacteria[lg(CFU/ml)](7.29±0.04)was also significantly lower than that in control group(7.93±0.15)(t=6.93,P=0.002).SEM observed that the bacteria decreased and the cell fragments appeared in DMAEM@RA group.In addition,DMAEM@RA significantly reduced the biomass of extracellular polysaccharides in the dual-species biofilm under acidic conditions.At pH=5.0,the biomass of extracellular polysaccharides in DMAEM@RA group[(25.13±3.14)mm3/mm2]was significantly lower than that in the control group[(42.66±7.46)mm3/mm2](t=3.75,P=0.020).DMAEM@RA could significantly up-regulate the expressions of gtfB and gtfC genes in Sm under acidic conditions.At pH=5.0,gtfB and gtfC genes were significantly up-regulated by(14.64± 0.44)times and(2.99±0.20)times,respectively(t=-42.74,P＜0.001;t=-13.55,P＜0.001).Conclusions The DMAEM@RA has a good antibacterial effect under acidic conditions,demonstrating that it has a good potential to prevent the occurrence and development of secondary caries.
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