【作者】 黄丽；

【导师】 樊明文；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2013， 博士

【摘要】 龋病是人类最常见的细菌感染性疾病之一,变异链球菌(Streptococcus mutans, S. mutans)是其主要的致病菌。变异链球菌作为牙菌斑的优势菌在菌斑的形成、发展和成熟过程中发挥重要作用。牙菌斑是龋病的始动因素,作为典型的细菌性生物膜,由链球菌属,放线菌属,乳杆菌属和其他菌属微生物细胞和细胞外多糖基质组成,各种细菌存在于细菌胞外多聚物基质包绕的立体三维结构中,相互黏附、附着、定植于牙表面及界面。变异链球菌产生的酶在糖代谢中起主导作用,产酸能力和耐酸性使之在菌斑酸化和釉质的脱矿中起作用。变异链球菌表面蛋白抗原PAc参与牙表面获得性膜的非选择性黏附,能够促进变异链球菌在牙面的定植和牙菌斑的形成,PAc在细菌对牙面的初始黏附中起重要作用,是致龋的重要毒力因子之一。变异链球菌另外一个重要毒力因子是GTFs,其葡聚糖结合区(glucan-binding domain,GLU)负责葡聚糖的合成。合成的葡聚糖介导了细菌和牙面之间的蔗糖依赖性黏附,作用是使得变异链球菌更加紧密地黏附于牙面,形成致密的牙菌斑。因此将PAc与GLU作为候选的抗原通过免疫学手段控制致病菌的感染,可以达到防治龋病的目的。本课题组成功构建了编码人细胞毒性T淋巴细胞的相关抗原4(cytotoxic T lymphocyte-associated antigen4, CTLA-4)的信号肽和胞外区基因,免疫球蛋白IgAl型的绞链区和CH2和CH3区基因片段,变异链球菌葡糖基转移酶葡聚糖结合区(GLU区)和表面蛋白抗原PAcA区和P区的靶向融合防龋DNA质粒pGJA-P,以FDA认可的PVAX1为载体构建新型的DNA疫苗pGJA-P/VAX,且证实可以有效激发唾液特异型IgA (secretory IgA,S-IgA)抗体的产生,龋齿积分结果也显示其确实存在明显的免疫防龋效果。本实验旨在证明pGJA-P/VAX1的免疫效果是否是因为其刺激唾液特异性IgA (secretory IgA, S-IgA)抗体的产生而影响了牙菌斑的形成,实验同时在大鼠体内外检测S-IgA对变异链球菌黏附力的影响机制。实验将靶向融合防龋DNA疫苗pGJA-P/VAX1免疫大鼠,观察诱导产生的特异型分泌型IgA(secretory IgA,S-IgA)抗体动力学；使用变异链球菌UA140::Φ(mutAp-mrfρ1)体外培养变异链球菌生物膜；使用特异型IgA(secretory IgA,S-IgA)抗体作用于不同时期的变异链球菌生物膜,激光共聚焦显微镜(confocal laser scanning microscope,CLSM)观察变异链球菌生物膜厚度的变化,CFU计数计算生物膜重悬细菌的数量以检测S-IgA抗体对变异链球菌的黏附能力的影响,同时探讨了S-IgA影响变异链球菌黏附能力的机制；实验还研究了DNA疫苗诱导产生的唾液分泌型IgA对变异链球菌在HA珠上的黏附的影响,得出唾液产生阻断变链黏附作用的最高稀释倍数；实验还同时在大鼠口腔内使用测量生物膜厚度,检测牙表面生物膜细菌量,扫描电镜观察生物膜细菌生长状态等多方面观察S-IgA对变异链球菌在动物体内牙面上黏附力的影响以及直观观察S-IgA在大鼠唾液中对变异链球菌生长状态的影响。本实验分为以下三个部分：第一部分：防龋DNA疫苗诱导产生唾液分泌型IgA对变异链球菌黏附力影响体外实验研究实验一靶向融合防龋DNA质粒pGJA-P/VAX1免疫原性和免疫效能的实验研究方法：20只6-8周龄雌性SD大鼠随机分为2组：靶向融合防龋DNA质粒pGJA-P/VAX1免疫组和空白载体pVAXl免疫组,将两种质粒分别与布比卡因(Bupivacaine)混旋,制备Bupivacaine-DNA复合体,经鼻粘膜滴注途径免疫SD大鼠,免疫前和免疫后每隔两周采取唾液至初免后16周,ELISA检测特异性IgA抗体的滴度水平。结果：经鼻粘膜滴注途径,pGJA-P/VAX1比PVAX1诱导了更高的唾液特异性IgA反应(p<0.01),且第10周达到高峰。结论：证实靶向融合防龋DNA质粒pGJA-P/VAX1可以有效诱导机体抗体反应。实验二体外变异链球菌生物膜模型的建立方法：使用UA140::Φ(mutAp-mrfρ1)体外培养变异链球菌生物膜。收集未免疫大鼠的空白唾液,60℃巴氏消毒30min,离心收集上清0.22μmm滤膜滤菌后备用。24孔板内置羟基磷灰石片,无菌唾液37℃摇床孵育4h形成获得性膜后去除唾液,每孔加BHI培养基,蔗糖,变异链球菌悬液厌氧共培养16h。细菌CFU计数使用振荡器上剧烈震荡2分钟的方法。取羟基磷灰石片,附着培养16h后生物膜的羟基磷灰石片,剧烈震荡后羟基磷灰石片各一片,常规处理扫描电镜观察。结果：HA片表面可以见到由规则六边形构成,最大程度模仿牙齿主要结构羟基磷灰石；16h后羟基磷灰石表面生物膜形成可以见到清晰的变异链球菌结构,变异链球菌在HA片表面均匀附着,形成生物膜结构,高倍镜下可以明显观察到链球菌的链状结构；振荡器剧烈震荡2min后表面存留变异链球菌很少,说明此重悬的方法能够有效去除HA表面附着的细菌,可以用于生物膜的后期重悬处理并用于细菌计数。结论：证实此方法可以用于本实验。实验三：S-IgA影响变异链球菌生物膜影响的机制的实验研究方法：使用不同的实验设计检测S-IgA减少变异链球菌生物膜形成的机制。Ⅰ组实验分别采用A组或B组唾液孵育4h在HA片上形成获得性膜后,然后加入正常未免疫大鼠培养生物膜,培养方法同前。实验结束,使用FITC标记的羊抗大鼠IgA标记获得性膜上的IgA,使用激光共聚焦显微镜观察获得性膜表面S-IgA的荧光强度是否有区别,并测量培养后的生物膜厚度和CFU计数,研究S-IgA是否对变异链球菌的初期定植产生影响；Ⅱ组实验采用正常唾液培养获得性膜后,加入A组或B组唾液与细菌共培养,观察对后期的黏附产生的影响。培养结束后取培养皿中剩余培养液,加入PBS稀释的FITC标记的羊抗大鼠IgAlml,使用PBS洗涤菌体数次,干净玻片涂片激光共聚焦显微镜观察S-IgA与变异链球菌是否存在结合情况;Ⅲ组实验常规培养变异链球菌生物膜16h后,在孔中加入A组或B组唾液,另设PBS对照组,37℃水浴孵育2h,观察S-IgA对成熟的生物膜是否产生影响。结果：使用A组唾液形成的获得性膜上面可以见S-IgA的附着且比B组明显增加,且形成生物膜厚度和CFU计数均与B组有显著性差异(P<0.01),A组形成获得性膜后在其上黏附的细菌厚度(少41.02%)和数量(少41.79)比B组形成的获得性膜要明显减少;直接使用两组唾液共培养,两组生物膜厚度和CFU存在显著性差异(P<0.01),A组形成的生物膜的厚度和黏附细菌数量分别比B组减少27.4%,22.81%,而且可以观察到S-IgA包裹在变异链球菌表而或是黏附在变异链球菌表面或同时产生作用；而对于牙菌斑生物膜已经形成,两组唾液对变异链球菌生物膜的厚度和CFU的影响无显著性差异(P>0.05);结论：S-IgA阻断变异链球菌生物膜的形成的机制在于可以参与形成获得性膜,对变异链球菌的初期黏附产生影响,且通过和变异链球菌表面成分相结合,促进变异链球菌的成团生长,影响其后期的定植,而对已经形成的生物膜没有明显作用。第二部分S-IgA对变异链球菌在羟基磷灰石珠上黏附力的影响研究实验四S-IgA对变异链球菌在羟基磷灰石珠上黏附力的影响结果方法：20只6-8周龄雌性SD大鼠随机分为2组：靶向融合防龋DNA质粒pGJA-P/VAX1免疫组和空白载体pVAX1免疫组,将两种质粒分别与布比卡因(Bupivacaine)混旋,制备Bupivacaine-DNA复合体,经鼻粘膜滴注途径免疫SD大鼠,免疫前和免疫后每隔两周采取唾液至初免后16周,ELISA检测特异性IgA抗体与唾液总IgA比值。取5mg羟基磷灰石检测分别与第4,10,16周大鼠唾液共培养1小时观察这几周唾液分别对变异链球菌的黏附力的影响。另外将第10周唾液分别稀释1：2；1：4；1：8；1：16；1：32后加入共培养1小时检测能发挥阻断作用的最大稀释倍数。培养结束后使用KCL洗涤三次,取洗涤后的悬浮菌液使用液体闪烁谱仪根据变异链球菌特性计算菌液中细菌量。其中使用对照组唾液和PBS作为对照组。结果：经鼻粘膜滴注途径,pGJA-P/VAX1比PVAX1诱导了更高的唾液特异性IgA反应(p<0.01),且第10周达到高峰;4w,10w,16w唾液处理后HA片上粘附的细菌量比对照组和PBS组都有明显减少(P<0.(01),且第十周唾液的阻断作用更加明显；10周唾液经过1：2；1：4；1：8；1：16稀释后与对照组和空白组相比均更多减少变异链球菌的黏附(P<0.01),且1：16是最高稀释倍数。结论：疫苗免疫后的大鼠唾液持续存在阻断变异链球菌在HA上的黏附且此黏附作用与唾液中S-IgA的量成正相关。第三部分防龋DNA疫苗诱导产生唾液分泌型IgA对变异链球菌黏附力影响的体内实验研究实验五S-IgA在大鼠口腔内对变异链球菌在牙而上黏附的影响方法：免疫后第九周开始一周内去除大鼠口腔内牙菌斑,最后一次清洁牙面4小时后断颈处死大鼠,无菌环境下取下带大鼠磨牙的下颌骨,体外使用Rhodamine标记的羊抗大鼠IGA二抗孵育(1：2500稀释于PBS中)2h后激光共聚焦显微镜观察表面S-IgA荧光强度的对比,观察S--IgA是否参与获得性膜的形成。之后两天无菌棉签定菌,48h后取大鼠唾液和上下颌磨牙：左上颌磨牙使用扫描电镜观察牙表面变异链球菌黏附细菌的量和形态的区别；左下颌的磨牙激光共聚焦显微镜观察牙表面变异链球菌黏附的量的比较；右上颌磨牙体外振荡表面黏附细菌后细菌计数；激光共聚焦显微镜观察唾液中S-IgA与变异链球菌相互作用的情况；并在定菌后持续取唾液样本检测唾液中UA140::Φ(mutAp-gfp1)总变链的比值。结果：实验组唾液比对照组唾液在牙表面形成获得性膜的荧光强度高,说明S-IgA参与获得性膜的形成；扫描电镜实验组牙表面变异链球菌比对照组细菌明显减少；实验组牙表面生物膜厚度和细菌量比对照组明显减少(P<0.01)；且在激光共聚焦显微镜下可见实验组唾液中的变异链球菌多成团生长,而对照组唾液中变异链球菌成散在分布,镜下可观察到S-IgA能够与变异链球菌紧密结合,包裹在细菌表面；实验期间实验组显著降低了定菌数口腔内UA140::Φ(mutAp-gfp1)占口腔变链的百分比(P<0.01),此趋势一直维持到实验结束。结论：防龋DNA疫苗诱导产生唾液分泌型IgA在体内也能明显减少变异链球菌对牙面的黏附,并能通过形成获得性膜和促进细菌的成团生长利于清除起到防龋的效果。更多还原

【Abstract】 Dental caries is one of the most common infectious diseases in the word. Streptococcus mutansfS.mutansJh the major etiological agent of dental caries. Plaque is the incipient factor of caries.As the typical bacteria biofilmjt is made up of Streptococci,Actino?nycetes,Lactobacilli,other microorganism cell and extracellular polysaccharides matrix. All kinds of bacteria exist in the three-dimensional structure which is surrounded by extracellular polymer substances matrix.They conglutinate,adhere and fix on the surface of teeth and other interfaces. As the preponderant bacteria of biofilm,S.mutans play important part in the formation、 development and muture of plaque.The enzyme of S.mutans has ruling action in the sugar metabolizing.It can produce acid and can live in acid environment, which have important impact in the acidification of plaque and the enamel demineralization. A cell surface protein antigen (PAc) mediate sucrose-independent adherence of S.mutans to tooth surface. PAc contribute to the adherence and accumulation of organism in dental plaque,so it is considered to be one of the important virulence factor of S.mutans. Another important virulence factor of it is GTFs,one region of which is glucan-binding domain((GLU).GLU is responsible for the composition of glucan. Glucans mediate sucrose-dependent adherence of S.mutans,making S.mutans adhere on tooth surface tightly and form compact dental plaque.An important approach to prevent dental caries would be consider PAc and GLU as the candidate antigen to control the infection of S.mutans by immune method. Our group have build the DNA plasmid pGJA-P which encode the signal peptide and extracellular regions of human CTLA4, CH2and CH3regions of IgA1,the GLU gene and A-P fragment of S.mutanspac gene. A new targeted anti-caries DNA plasmid pGJA-P/VAXl was successfully constructed join the pGJA-P with the pVAXl,the only vector authorized by Food and Drug Administration in clinical trials. It is proved that the vaccine can stimulate the secretion of IgA (secretory IgA,S-IgA) in saliva and the results of caries scores also prove the truth the DNA vaccine has the effect of anti-caries.The purpose of this study is to detect whether the immunity effect of pGJA-P/VAXl is that the S-IgA antibody interfere the formation of dental plaque. Study the kinetics of the antibody responses generated following targeted anti-caries DNA plasmid immunization.Use the S-IgA to influence the biofilm cultivated by UA140:: Φ(mutAp-rfpI),then use the CLSM to determine the depth of biofilm and CFU to determine the effect of S-IgA on the adherence ratio of S.mutans and the possible mechanism of the effect at the same time;the effect of S-IgA antibodies on the adhesion of S. mutas MT8148onto S-HA beads was examined;in additional,we study inhibition of Streptococcus mutans biofilm formation by S-IgA antibodies induced by DNA vaccine in vivo through measure the depth of biofilm, calculating the adherence bacterial number by CFU and observing the growth condition of S.mutans on tooth of rats by CLSM.The present study consists of three parts:Part one: Inhibition of Streptococcus mutans biofilm formation by secretory immunoglobulin A (S-IgA) antibodies induced by targeted fusion anti-caries DNA vaccine in vitro.Experiment Ⅰ: Kinetics of the antibody responses generated following targeted anti-caries DNA plasmid immunization.Method:Four-to-six-week-old female Wistar rats,8per group, were immunized with pGJA-P/VAX (Group A) or pVAXl (Group B) by the intranasal route at week0and week2, respectively.Bupivacaine:DNA complexes were prepared by adding bupivacaine hydrochloride to the aqueous DNA solution using a fast mixing method. SD rats were immunized with Bupivacaine:pGJA-P/VAX1complex or Bupivacaine:pVAXl complex intranasally. Saliva samples were collected for ELISA.Result:The salivary IgA response of pGJA-P/VAX1i.n. immunized group were significantly higher than those of the pVAXl i.n. immunized group (p<0.01) and the level of SIgA arrived climax in10weeks after first immunization. Conclusion: The targeted fusion DNA vaccine pGJA-P/VAX1markedly enhance the magnitude of mucosal specific antibody response via the intranasal route. Expriment II:Preparation of S. mutans bio filmsMethod:HA discs were placed in the wells of24-well polystyrene cell culture plate and incubated under agitation for4hours at37℃with0.5ml of sterile saliva. After that,the saliva were removed and replaced with0.8ml of TSB+0.15%(w/v) sucrose and0.8ml of sterile saliva.Each well was inoculated at37℃with S.mutans UA140::Φ(mufAp-rfp1)0.2ml and culture plates were incubated aerobically at37℃for16h,the HA discs were dip-washed twice in the PBS and swirled gently to remove loosely adherent bacteria to harvest the adherent cells.each disc were transferred to a sterile50-ml polypropylene tube containing1ml of PBS and vortexed vigorously for2min.Viable counts were carried by this dilusion.SEM was used to determine(ⅰ) the surface of HA disc,(ⅱ)the formation of bacterial biofilms,(ⅲ)the efficacy of the resuspension of protocol.Result:saliva pellicle was formed evenly on the HA disc bacterial biofilms can be easily observed under the SEM,the resuspension of protocol was satisfactory because few bacterial can be seen on the HA disc and can be used for CFU.ConclusioniThis method can be used in our experiment. Experiment Ⅲ:The mechanism of the effect of S-IgA on the adherence of S.mutans onto HA diskMethod:Use different designs to find out the mechanism of the effect of S-IgA on S.mutans biofilm. group Ⅰ:acquired pellicle was formed by saliva from groups A and B under agitation, replaced with BHI, sucrose, S. mutans and sterile blank saliva,biofilms were grown for16h before the cells were harvested and enumerated, and the depth of the biofilms was measured by confocal; group II:acquired pellicles were formed on HA disks with blank saliva,then the saliva was replaced with BHI, sucrose, S. mutans and sterile saliva from experimental or control groups.S.mutans biofilms were cultivated for16h, cells were recovered and enumerate;groupIII:S. mutans biofilms were cultivated with blank saliva for16h transferred to new wells containing sterile saliva from experimental or control groups, viable bacteria counts and biofilm measurement were carried out. Result:More S-IgA could be seen from the pellicle formed by saliva from group A compared with the pellicle formed by saliva from group B, there were significant differences in the number of cells recovered from the biofilm(41.79%lesser) and the depth of the biofilm (41.02%lesser)(P<0.01); bacteria count obtained from HA disks (27.4%less) and measured in the experimental group (22.81%less) were less than biofilms obtained in the control group (P<0.01); S-IgA could assemble S. mutans together or cover the surface of S.mutans; while there is no significant differences between treatment on mature biofilm(P>0.05). Conclusion:the mechanism of effect of S-IgA on biofilm is to effect the early adherence of S.mutans by forming plaque;and prevent the adherence of S.mutans later by joining with protein on the surface of S.mutans,while have no effect on mature bioflimPart Two:The effect of S-IgA antibodies on the adhesion of S. mutans MT8148onto HA beadsExperiment Ⅳ:The effect of S-IgA antibodies on the adhesion of S. mutans MT8148onto S-HA beadsMethod:Four-to-six-week-old female Wistar rats,8per group, were immunized with pGJA-P/VAX (Group A) or pVAX1(Group B) by the intranasal route at week0and week2, respectively.Bupivacaine:DNA complexes were prepared by adding bupivacaine hydrochloride to the aqueous DNA solution using a fast mixing method. Saliva samples were collected at2,4,6,8,10,12,14, and16weeks after the first immunization and ELISA to measure SIgA/total TgA.5mg of HA beads were equilibrated in buffered KC1and cultivated with4-week,10-week, and16-week saliva, and serial dilutions (1:2;1:4;1:8;1:16;1:32) of10-week saliva of immunized mouse and unimmunized mouse and PBS was used as control. Result:The IgA anti-Pac and anti-Glu responses in rats peaked at10weeks, The adherence of S. mutans in saliva of experiment rats was significantly lower than that in the control group (p<0.001). The S-IgA antibodies in10week has the strongest inhibition of the adhesion of S. mutans cells, but there were no significant differences between the4th,10th, and16th weeks, the lowest dilution of saliva with effect was1:16, and reduced the adherence activity of S. mutans to approximately8.7%.Conchision:the salivary sample containing specific S-IgA has the consistent effect through out the experiment and also shown to inhibit the binding of S. mutansin a dose-dependent manner.Part Three:Inhibition of Streptococcus mutans biofilm formation by secretory immunoglobulin A (S-IgA) antibodies induced by targeted fusion anti-caries DNA vaccine in vivoExperiment V:Inhibition of Streptococcus mutans biofilm formation by secretory immunoglobulin A (S-IgA) antibodies induced by targeted fusion anti-caries DNA vaccine in vivo.Method:At the9th week after immunization, remove the biofilm on the surfaces of tooth of rats by mechanism methods, broken neck death after4h behind the last cleaing, removed the mandibular molars of rats,observe the fluorescence intensity on the tooth after co-cultivated the tooth with anti-RAT IgA antibody (Rhodamine Conjugated) by confocal laser microscope to observe whether SIgA is involved in the formation of the acquired membrane.48h after putting S.mutans onti mouth of rats,saliva and mandibular molar were collected;S. mutans adhesion amount were observes on the left maxillary molar using scanning electron microscopy; the comparison of the amount of S. mutans adhesion on tooth surface were taken from the left mandibular molars by confocal laser microscope; bacterial count were carried out by right maxillary molar oscillation surface; S-IgA in saliva and S. mutans adhesion were observed by confocal; and testing UA140::Φ(mutAp-gfp1)/total Streptococcus in saliva Results:fluorescence intensity in the experimental group is higher than control group,showing SlgA involved in the formation of the acquired membrane; scanning electron microscopy (SEM)showed experimental tooth surface had more S. mutans bacteria than the control group; there is significant differences between the biofilm thickness and bacterial amount in the control group and experimental group (P<0.01); and S. mutans clouds grow more in the saliva in the experimental group whereas the S. mutans in saliva of control group grown as scattered distribution; S-IgA can closely combine with S. mutans,wrapped in the surface of the bacteria; During the experiment the experimental group significantly reduced the percentage of UA140::Φ(mutAp-gfpI)/total Streptococcus (P<0.01),and the trend has been maintained to the end of the experiment.Conclusion: targeted fusion anti-caries DNA vaccine could induce the production of S-IgA and can be significantly reduced adhesion of Streptococcus mutans on the tooth surface in vIvo.更多还原