变异链球菌反义vicK RNA对口腔链球菌多菌种生物膜致龋性的抑制作用  

Inhibitory effect of Streptococcus mutans antisense vicK RNA regulating the cariogenicity of oral streptococci multi-species biofilm

作　　者：陈虹 许萌萌 孙雨婷 余霜 杨德琴[1] Chen Hong;Xu Mengmeng;Sun Yuting;Yu Shuang;Yang Deqin(Department of Endodontics,Stomatological Hospital of Chongqing Medical University&Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences&Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education,Chongqing 401147,China)

机构地区：[1]重庆医科大学附属口腔医院北部院区牙体牙髓科、口腔疾病与生物医学重庆市重点实验室、重庆市高校市级口腔生物医学工程重点实验室,重庆401147

出　　处：《中华口腔医学杂志》2024年第1期64-70,共7页Chinese Journal of Stomatology

基　　金：国家自然科学基金(82100991);中国博士后科学基金(2021M700627);重庆市自然科学基金博士后科学基金(cstc2021jcyj-bshX0211);重庆市渝中区科学技术局基础研究与前沿探索项目(20210124);重庆市博士后资助项目(2010010005994583);重庆市教委科学技术研究计划青年项目(KJQN202100440)。

摘　　要：目的研究变异链球菌(Sm)反义vicK RNA(ASvicK)对3种常见口腔链球菌[Sm、血链球菌(Ss)和戈登链球菌(Sg)]构建的多菌种生物膜致龋性的调控作用。方法通过重组质粒构建ASvicK过表达株,以Sm标准菌株UA159和ASvicK过表达株分别与Ss及Sg构建的多菌种生物膜(分别为UA159+Ss+Sg组、ASvicK+Ss+Sg组)为研究对象,扫描电镜观察生物膜结构;结晶紫染色法检测细菌生物膜总量的差异;通过乳酸试剂盒及蒽酮法评估生物膜产酸产糖能力;利用TaqMan荧光定量PCR及实时荧光定量PCR检测生物膜中3种菌的比例及Sm致龋相关基因的改变;进一步构建人牙釉质片生物膜脱矿模型,用显微硬度仪检测牙釉质表面硬度变化。结果 ASvicK过表达后,Sm+Ss+Sg多菌种生物膜结构疏松。相较于UA159+Ss+Sg组,ASvicK+Ss+Sg组生物膜总量、乳酸产量分别显著降低78.93%及62.23%(均P<0.001),而生物膜水不溶性和不溶性胞外多糖产量分别显著降低39.13%及68.00%(均P<0.001)。与UA159+Ss+Sg组相比,ASvicK+Ss+Sg多菌种生物膜中致龋菌Sm比例显著降低33.00%(P<0.01),Sm致龋相关基因vicK/X、gtfB、gtfC、gtfD和ftf表达显著下调(P<0.05),牙釉质片脱矿后显微硬度值显著上升至183.84%(P<0.001)。结论 ASvicK过表达可降低口腔链球菌生物膜中致龋菌Sm的比例,并减弱口腔链球菌生物膜致龋毒力及致龋性,可能减缓龋病的进展。Objective To investigate the regulative effects of Streptococcus mutans(Sm)antisense vicK RNA(ASvicK)on the multi-species biofilm formed by three common oral streptococci(Sm,Streptococcus sanguinis,and Streptococcus gordonii)(Sm+Ss+Sg).Methods ASvicK over-expression strain was constructed by using a recombinant plasmid,and three-species biofilm UA159+Ss+Sg and ASvicK+Ss+Sg were cultured.The phenotypes of biofilms were detected by scanning electron microscopy(SEM).Crystal violet(CV)assay was used to detect biofilm biomass.Lactate kit and anthrone-sulfuric acid colorimetric assay were used to determine the abilities of lactic acid and exopolysaccharides production,respectively.The proportions of three-species and expression levels of the cariogenic-related genes in biofilms were detected by TaqMan fluorescence quantitative PCR and real-time fluorescence quantitative PCR.A biofilm demineralization model of human enamel slabs was further constructed,and the hardness of enamel surface was detected.Results Compared to UA159+Ss+Sg,over-expression of ASvicK could inhibit biofilm formation and lactic acid production in ASvicK+Ss+Sg biofilm significantly decreased by 78.93%(P<0.001)and 62.23%(P<0.001),respectively.With ASvicK over-expression,the amounts of water-insoluble and-soluble glucoses in ASvicK+Ss+Sg biofilm were reduced respectively by 39.13%(P<0.001)and 68.00%(P<0.001).Compared to the UA159+Ss+Sg Group,the proportion of Sm,the cariogenic bacteria,showed 33.00%reduction(P<0.01)in Sm+Ss+Sg biofilm,and the gene expressions of cariogenic-relative genes vicK/X,gtfB/C/D,and ftf significantly decreased(P<0.05).The micro-hardness value of enamel slabs after demineralization by ASvicK+Ss+Sg biofilm was significantly increased to 183.84%(P<0.001).Conclusions ASvicK over-expression could reduce the Sm proportion and weaken the cariogenicity of oral Streptococcus biofilm,thereby possibly slowing down the progression of caries.

关 键 词：龋齿 链球菌 变异 反义vicK RNA 血链球菌 戈登链球菌 

分 类 号：R781.1[医药卫生—口腔医学]