【作者】 张蕾；

【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】 龋病是一种由口腔中多种因素复合作用所导致的牙体硬组织进行性病损,大量临床及流行病学调查结果显示,中国居民各年龄段的患龋率均居高不下。龋病中细菌因素为主要的致龋因素,变形链球菌为主要致龋菌之一。致龋菌在口腔内的产酸代谢活动是龋病产生的直接原因。因此,有效的干扰致龋菌的生长和代谢,即可达到预防控制龋病的目的。针对目前防龋方法的缺陷,探索一种安全有效的新型防龋方法显得尤为重要。光动力疗法(Photodynamic therapy, PDT)是一种有效的抗微生物疗法,其灭活细菌的作用受到越来越多的关注,它可有效治疗细菌性疾病而不产生耐药性,有利于在龋病预防中的应用。国内外已有应用PDT方法作用于体外游离菌及人工龋口腔生物膜菌斑中变形链球菌的研究,取得了良好的杀伤效果。本课题就是通过优化、选择合适的PDT参数,在构建的大鼠龋齿模型上探讨PDT的防龋作用及安全性。目的：通过应用532nm和630nm的半导体激光器和光敏剂血卟啉单甲醚(Hematoporyrin monomethyl Ether, HMME)对体外培养的变形链球菌(Streptococcus mutans, S.mutans)进行光动力治疗,优化光敏剂孵育时间、浓度,选择适宜波长的激光器和激光照射参数；建立大鼠龋齿模型并进行光动力实验,从而探讨不同能量密度的PDT对大鼠口腔中S.mutans的杀伤作用、对龋齿的预防作用以及对口腔软组织安全性的影响,为PDT防龋的临床实践提供理论与实验依据。方法：1.将S.mutans于37℃恒温培养箱中复苏,厌氧培养24h,制备1×108CFU/m1标准菌液待用。2.应用532nm和630nm的半导体激光器和光敏剂HMME对96孔板内的S.mutans菌液进行PDT体外实验,根据平板菌落计数的结果,选择适宜波长的激光器、光敏剂孵育时间和激光参数,为下一步的大鼠龋齿模型实验提供依据。3.通过在大鼠口腔接种标准S.mutans菌液,并喂以致龋饲料2000#,构建大鼠龋齿模型。4.将56只Wistar大鼠随机分成七组：能量密度8.4J/cm2PDT组、能量密度12.6J/cm2pDT组、能量密度16.8J/cm2pDT组和单纯光敏剂组、单纯激光组、0.2%(质量分数)NaF阳性对照组、0.9%(质量分数)生理盐水阴性对照组。选择波长532nm,功率密度140mw/cm2的半导体激光为光源,40μg/ml HMME为光敏剂,按照上述分组进行PDT处理,每周进行一次,取样进行平板菌落计数,直至56d实验结束。依照平板菌落计数法观察各组对患龋牙面上S.mutans的杀伤作用；通过Keyes记分观察龋齿预防效果；通过组织病理观察对大鼠口腔软组织的影响。结果：1.体外实验光动力照射S.mutans 96孔板的平板菌落计数结果显示,本实验最适合的PDT组合为光敏剂HMME浓度40μg/m1、孵育时间5min,半导体激光波长532nm、功率密度140mw/cm2。2.根据HMME-PDT对致龋模型中S.mutans杀伤作用的平板菌落计数显示,与0.9%生理盐水阴性对照组相比,单纯HMME组S.mutans数量无显著差异性(P>0.05)；单纯激光照射组和0.2%NaF阳性对照组,均能使S.mutans数量(CFU/ml)减少(P<0.05),杀菌率为50.38%、38.93%；而PDT防龋处理各组均可使大鼠致龋模型口腔中S. mutans数量显著减少(P<0.05),能量密度8.4J/cm2、12.6J/cm2、16.8J/cm2pDT组的杀菌率分别为61.49%、96.91%、97.52%。其中能量密度12.6J/cm2、16.8J/cm2pDT组与能量密度8.4J/cm2pDT组相比杀菌率显著提高(P<0.05),而能量密度12.6J/cm2pDT组与能量密度16.8J/cm2pDT组之间没有显著性差异(P>0.05)。3.在体视显微镜下对大鼠磨牙咬合面窝沟龋齿分别进行Keyes记分。由Keyes记分结果可以看出,与0.9%生理盐水阴性对照组相比,PDT处理组、NaF组、单纯激光组均有统计学意义(P<0.05),单纯光敏剂组无统计学意义(P>0.05)。各PDT处理组之间相比,随着能量密度的不断增加,Keyes记分呈现降低的趋势,但彼此之间并无统计学意义(P<0.05)。4.与空白对照组比较,各PDT防龋处理组的口腔软组织样本,都出现不同程度的改变。能量密度8.4J/cm2pDT组和12.6J/cm2pDT组中颊、舌粘膜充血、水肿现象的例数均少于16.8J/cm2pDT组；16.8J/cm2pDT组中,烦、舌粘膜充血水肿例数明显增多,也出现了少数轻度炎症细胞浸润现象,偶有牙龈出现充血、角化层变厚现象,与总体样本数比较,充血和水肿现象是主要的组织病理反应。结论：1.HMME-PDT对体外S.mutans有明显的杀伤作用,杀伤效果与光敏剂的孵育时间、浓度、激光照射时间和功率密度相关。2.HMME-PDT对大鼠龋齿有明显的预防作用,其预防效果与能量密度关系密切。3.不同能量密度的PDT激光照射牙齿,会造成口腔软组织不同程度的改变,PDT激光能量密度越大,越容易造成口腔软组织的充血,水肿等病理改变。在后续实验和临床应用中应选择较为理想的能量密度,以降低口腔软组织的损伤。更多还原

【Abstract】 Dental caries is the progressive diseases in dental hard tissue caused by a variety of complex factors in oral cavity, a large number of clinical and epidemiological surveys show that the dental caries prevalence rate of all ages in Chinese remains high for many years. Bacterial factor is the major cariogenic factor in dental caries, Streptococcus mutans is one of the main cariogenic bacteria. The direct reason of dental caries is the acid metabolic activity caused by Cariogenic bacteria in the mouth. Therefore, we can achieve the purpose of preventing and controlling caries by interfering growth and metabolism of cariogenic bacteria effectively. Aiming at the defect of preventing dental caries, to explore a safe and effective new method of dental caries prevention is very important.Photodynamic therapy (PDT) is an effective antimicrobial therapy, more and more attention of its effect on inactivating bacteria are attracted. It can treat bacterial diseases effectively without drug resistance and in favor of the prevention of dental caries. The domestic and foreign scholars methods have applied PDT on Streptococcus mutans in vitro and oral biofilm in artificial caries, and achieved a good antibacterial effects. The topic of this subject is to discuss the prevention and safety of dental caries in the rat caries model of PDT by the optimizing and selecting the appropriate parameters of PDT.Objective:Using HMME-PDT to kill S. mutans in vitro by the semiconductor laser with the wave of 532nm and 630nm respectively.Optimizing photosensitizer incubation time, photosensitizer concentration and various laser parameters to choose the best laser, photosensitizer incubation time and laser exposure parameters. Establishing rats caries model for PDT, to investigate the killing effect on S.mutans in rats oral cavity and the preventive effect on caries and the safety of oral soft tissue by PDT of different energy densities, providing the theoretical and experimental basis on PDT clinical practice.Methods: 1. S.mutans was recovered in the standing-temperature cultivator at 37℃and anaerobic cultured for 24h, and prepared concentration of standard bacteria solution with 1×108CFU/ml and set aside.2. S.mutans in 96 pores plate was treated with HMME-PDT by the semiconductor laser with the wave of 532nm and 630nm. Chose the appropriate laser,photosensitizer incubation time and various laser parameters, to provide the basis of rats caries experiment according to plate count results.3. Establishing rats caries model by inoculating tandard solution of S.mutans in the rat oral cavity and feeding diet 2000#4.Dividing 56 Wistar rats into seven groups randomly:energy density of 8.4J/cm2、12.6J/cm2、16.8J/cm2 PDT groups and the photosensitizer group, simple laser group,0.2% NaF positive control group,0.9% normal saline negative control group. Elected the semiconductor laser with wavelength 532nm and power density 140mw/cm2 as light source,40μg/ml HMME as photosensitizer for PDT in accordance with the above groups weekly. Sampled the plate colony count until 56d at the end of the experiment. Observed the killing effect of S.mutanss in dental caries each group according the result of plate colony count; observed the caries preventive effect by Keyes points; observed the oral soft tissue in rats by histopathologic examination.Results:1.The colony counts of S.mutans in 96 plates which was treated with PDT in vitro showed that the best combination of this experiment with photosensitizer concentration 40μg/ml of HMME, incubation time 5min, the semiconductor laser of wavelength of 532nm and power density 140mw/cm2.2.Plate colony count of the inhibition of HMME-PDT with different energy density on S.mutans in rats showed that compared with 0.9% saline negative control group, the difference of S.mutans in HMME group was no significant (P＞0.05); the number of S.mutans (CFU/ml) decreased (P＜0.05) in both laser irradiation group and 0.2% NaF group,sterilization were 50.38% and 38.93%;the number of S.mutans was reduced significantly (P<0.05);in every PDT treatment groups, the sterilization rate in energy density of 8.4J/cm2、12.6J/cm2、16.8J/cm2 PDT group were 65.06%,96.97%, 97.52% respectively. The sterilization rate of 12.6J/cm2and 16.8J/cm2 PDT group increased significantly (P＜0.05) compared to the 8.4J/cm2 PDT group, while there was no significant difference between the 12.6J/cm2 and 16.8J/cm2 PDT group (P＞0.05).3. Keyes points results of fissures in rats under the stereomicroscope showed that compared with the 0.9% NS negative control group the difference in the experimental group, the NaF group and the laser group were significant statistically (P＜0.05), the difference in the photosensitizer group was not significant(P＞0.05). The score decreased by the increasing energy density in each PDT groups which were compared with each other, but the difference was not significant(P＞0.05).4. There were changes of cheek, tongue mucosa, gingival mucosa samples in every PDT groups compared with the blank control group in varying degrees. The number of congestion, edema of buccal, lingual mucosa cases in 8.4J/cm2 and 12.6J/cm2 PDT group were less than 16.8J/cm2 PDT group. The number of these cases in 16.8J/cm2 PDT group increased significantly; there have been slight infiltration of inflammatory cells in few samples.The phenomenon of thickened keratinized layer and bleeding in gum appeared occasionally. The phenomenon of congestion and edema were the main pathological response in all samples. Conclusion:1.The inhibitiont of HMME-PDT on S.mutans in vitro was significantly. The sterilizing result was related with the photosensitizer incubation time、concentration and the exposure time and power density.2. The prevention of HMME-PDT on rat dental caries significantly, but the effects were related to the energy density closely.3. Different energy density of PDT can cause different degrees of changes of oral soft tissues. With the energy density increasing, the pathological changes of congestion, edema in oral soft tissues and gums occurred easily. We should choose the ideal energy density of PDT in the follow-up experimental and clinical application to reduce the damage of oral soft tissue.更多还原