【作者】 李敏；

【导师】 邹朝晖；

【作者基本信息】 天津医科大学 ， 口腔临床医学， 2011， 硕士

【摘要】 龋病是牙齿在外界因素影响下,牙齿的釉质、牙本质或牙骨质发生的一种进行性破坏性疾病,其本质是一种细菌感染性疾病。龋病产生的直接原因是牙菌斑内细菌的产酸代谢活动,牙菌斑中含有许多具有致龋作用的细菌,如变形链球菌(Streptococcus mutans, S.mutans)、黏性放线菌、血链球菌、乳杆菌等,目前已经公认龋病的主要致龋菌为变形链球菌,因此,通过适当的方法抑制龋病的主要致龋菌—变形链球菌生长,是预防龋病的重要途径之一。通过体外实验,结果表明激光波长为532nm,血卟啉单甲醚(hematoporphyrin monomethylether, HMME)为光敏剂的光动力疗法(photodynamic therapy, PDT)对变形链球菌有良好的杀伤效果,为防龋的研究开辟了一种新途径。为进一步研究PDT对口腔内变形链球菌的影响,又建立了大鼠龋病模型。在建模期间,给予不同浓度的HMME-PDT处理,每周一次,连续四周,记录变形链球菌在大鼠口腔内的生长情况及观察PDT对大鼠龋病的发生、发展的影响,并比较其防龋效果与氟化钠防龋效果的差别,初步探讨合适浓度的HMME的光动力效应在龋病预防中的作用。目的：①探讨不同浓度参数的血卟啉单甲醚(HMME),在波长为532nm激光作用下,对体外变形链球菌的影响,为动物实验研究做准备。②建立龋病模型探讨不同浓度的HMME-PDT对大鼠口腔内变形链球菌的生长及龋病发生的影响,寻找合适的光敏剂剂量,为将PDT用于临床上预防龋病提供理论基础和实验依据。方法：①变形链球菌菌液分别与浓度为5μg/ml、10μg/ml、20μg/ml、40μg/ml、50μg/ml、60μg/ml的HMME孵育5min后,应用532nm半导体激光器照射(功率为120mw,功率密度为140mw/cm2),然后对菌液进行菌落计数,并与单纯光敏剂组、单纯激光组及空白对照组的菌落数进行比较,观察变形链球菌的活菌数。②采用变形链球菌接种Wistar大鼠磨牙,给予致龋饲料,建立龋病模型。将大鼠分为5组,分别用10μg/ml HMME-PDT(A组)、20μg/ml HMME-PDT(B组)、40μg/ml HMME-PDT (C组)、0.2g/L氟化钠水溶液(D组,阳性对照)和0.9%的生理盐水(E组,阴性对照)处理各组大鼠磨牙,每周1次,连续4周,记录大鼠口腔内变形链球菌的生长情况。处死大鼠后,用扫描电镜观察各组磨牙龋损区的表面形态和测定大鼠牙颊舌侧釉质显微硬度值。结果：①体外实验表明不同浓度的HMME-PDT对变形链球菌处理后,变形链球菌菌落数明显减少(P<0.05),且HMME浓度在一定范围内(5μg/ml～40μg/ml),随着HMME浓度的升高,变形链球菌菌落数逐渐下降；超过此范围PDT的杀菌效果未见明显增强。②动物实验表明第一、二周各组菌落数差异均无统计学意义(P>0.05),第三周光敏剂浓度为20μg/ml、40μg/ml时菌落数明显低于生理盐水组(P＜0.05)。第四周光敏剂浓度为10μg/ml、20、40μg/ml时菌落数均明显低于生理盐水组(P<0.05或P<0.01),其中40μg/ml HMME-PDT组的菌落数明显少于10μg/ml HMME-PDT组和20μg/ml HMME-PDT组(P<0.05)。③扫描电镜观察发现,40μg/ml HMME-PDT组与氟化钠组龋损破坏程度最轻且表面相对平滑。④显微硬度值结果表明：20μg/ml、40μg/mlHMME-PDT组和氟化钠组三组的牙颊侧釉质硬度值明显高于生理盐水组,且差异有统计学意义(P<0.05),但三组之间差异无统计学意义(P＞0.05)。各组舌侧牙釉质硬度值与生理盐水组相比,差异均无统计学意义(P>0.05)。结论：体外实验表明单纯激光或单纯光敏剂基本上对变形链球菌没有杀菌作用,而PDT对变形链球菌有明显的杀菌作用；此外PDT中的HMME浓度在一定范围内(5μg/ml-40μg/ml),随着HMME浓度的升高,变形链球菌菌落数逐渐下降；但超过此范围PDT的杀菌效果未见明显增强。大鼠龋病模型的建立,模拟了人类龋病的发展,用于PDT防龋的实验研究,40μg/mHMME-PDT能有效的杀灭大鼠口腔内的变形链球菌；PDT能够清洁牙面,控制牙菌斑在牙面的附着,同时减轻龋损的破坏程度；此外PDT能提高大鼠牙齿的表面硬度,减缓或阻止龋病的发展。所以HMME-PDT将会为龋病的预防提供一种新的有效的途径。更多还原

【Abstract】 Dental caries is a progressive disease influenced by external factors which damages the enamel, dentin or cementum. Its essence is a bacterial infectious disease. The formation of acid products through the metabolism activity within dental plague is a directed factor in the development of dental caries. Dental plague contains many cariogenic bacterium, for example, Streptococcus mutans (S.mutans), Actinomyces viscous, Streptococcus sangius and Lactobacilli has been currently implicated as the primary aetiological agent. So to inhibit S.mutans growth by appropriate method is an important path to the prevention of dental caries. The result of experiment in vivo shew that HMME-mediated photodynamic therapy can inhibit S.mutans effectively. PDT is thought to be positive in the protection of dental caries. In the period of establishing the caries rat model for further study, the rat’s teeth were treated with different concentrations of HMME-mediated PDT once a week for four weeks, recorded the changes of S.mutans and observed the effects of PDT on the development of dental caries. The anticaries result of PDT was compared with sodium fluoride. The experiment was preliminary discussed the proper concentration of HMME photodynamic effect in the role of caries prevention.Objectives:①To investigate the effects of different concentrations of HMME with the laser had the wavelength of 532nm on the growth of S. mutants in vitro, and prepared for the fllowing animal experiment.②To study the effect of different concentrations of HMME-mediated PDT on the growth of primary cariogenic S.mutants and the development of caries in rats, then find the suitable dose of photosensitizer. It will be provided theoretical basis and experimental evidence for PDT used for the prevention of caries in clinical.Methods:①The suspension of Streptococcus mutans incubated with different concentrations(5μg/ml、10μg/ml、20μg/ml、40μg/ml、50μg/ml、60μg/ml) of HMME for five minutes, then irradiated by the semiconductor laser whose wavelength is 532nm and output power was 120mw and the density power was 140mw/cm2. Finally, the viable counts of S. mutans recorded by counting colony forming unit were compared among the HMME-PDT groups, photosensitizer-only group, laser-only group, and blank control group. ①Wistar rats, whose molars were inoculated with Streptococcus mutans (S. mutans), were fed with caries-inducing diet 2000 in order to establish the caries rat model. These rats were divided into five groups, whose molars were treated respectively with 10μg/ml HMME-PDT (group A),20μg/ml HMME-PDT (group B), 40μg/ml HMME-PDT (group C),0.2 g/L sodium fluoride as positive controls (group D), and with 0.9% sterile physiological saline as negative controls (group E) once a week for four weeks and recorded the changes of S. mutans every week. The rats were then killed. The surface morphology of dental caries of the rats was observed by scanning electron microscope.Result:①The experiment in vitro shows that the viable counts of Streptococcus mutans of different consentrations of HMME-PDT are reduced significantly(P<0.05). The counts are reduced gradully with HMME concentration increasing from 5μg/ml to 40μg/ml, but there were no significant decreasing trends found when the HMME concentration continued increasing.②Animal experiment shows that the colony count in each group shows no significant difference in the first and second weeks (P>0.05). The colony counts of group 20μg/ml HMME-PDT and 40μg/ml HMME-PDT are significantly reduced compared with group 0.9% sterile physiological saline in the third week (P<0.05). In the fourth week the colony counts of group 10μg/ml、20μg/ml and 40μg/ml HMME-PDT are significantly reduced compared with group 0.9% sterile physiological saline (P<0.05 or P<0.01). Meanwhile the colony count of group 40μg/ml HMME-PDT is significantly lower than both group 10μg/ml and 20μg/ml HMME-PDT(P<0.05).③The tooth enamel of both group 40μg/ml HMME-PDT and 0.2g/L sodium fluoride are found to be damaged most slightly, and the tooth surfaces of the two groups are relatively smooth.④The hardness of buccal enamal of groups 20μg/ml、40μg/ml HMME-PDT and 0.2g/L sodium fluoride have no significant difference (P<0.05), but are significantly high compared with group of 0.9% sterile physiological saline (P<0.05). The hardness of lingual enamal in each group shows no significant difference (P<0.05).Conclusion:In vitro, the experiment shows that only the presence of photosensitizer or only the presence of the laser is almost no killing effect on Streptococcus mutans, but the combination of the two factors has a significant effect on Streptococcus mutans. The viable counts of Streptococcus mutans are reduced gradully between 5μg/ml HMME and 40μg/ml HMME. But over this range the effect of PDT on bactericidal was no significantly increased. The rat caries model simulating the developing of human dental caries is used for the experiment study of PDT. The group of 40μg/ml HMME-PDT kills Streptococcus mutans of rat oral effectlively. PDT can clean the tooth surface and control the dental plague attaching to tooth surface, meanwhile reducing the extent of the damage, so PDT is the key of anti-caries. In addition, PDT can improve the surface hardness of the rat’s tooth, that can slow or stop the process of dental caries. HMME-PDT will provide a new effective method for the prevention of dental caries.更多还原