【作者】 邹朝晖；

【导师】 李迎新； 高平；

【作者基本信息】 天津医科大学 ， 生物医学工程， 2009， 博士

【摘要】 龋病是危害人类健康的第三类疾病，它对健康的危害仅次于心血管疾病和肿瘤。变形链球菌等致龋微生物在口腔生物膜内形成牙菌斑，并进行产酸代谢活动是产生龋病的直接原因。因此，通过适当方法影响口腔生物膜内的细菌代谢，干扰牙菌斑的形成或促进牙菌斑的解离，即可达到预防龋病的目的。现有防龋方法中氟化物防龋法虽已得到普遍公认，但氟化物使用的安全性问题和变形链球菌耐氟菌株的出现都为预防龋病提出了新的课题。寻找更为安全有效的防龋方法是目前急需解决的问题。光动力疗法(Photodynamic Therapy，PDT)目前已成为肿瘤的基本治疗手段之一；近年来，在非肿瘤性疾病特别是细菌性疾病和病毒性疾病的治疗上，PDT也取得了显著的疗效。鉴于目前国外已有实验室开展PDT对单一口腔致龋菌的实验研究，但未见在人工龋模型上的实验研究，本研究从龋病预防的角度出发，探讨PDT对人工龋口腔生物膜致龋变形链球菌的作用。目的：构建符合人体生理环境的人工龋模型，并将其应用于PDT防龋实验；探讨PDT对口腔生物膜中主要致龋菌(变形链球菌)的作用；分析PDT过程中不同剂量的光敏剂对抑菌效果的影响，通过实验选择适宜预防龋病的激光能量剂量；结合DiO、Hoechest33342两种荧光探针在细胞水平上探讨PDT的防龋机制；为PDT防龋的临床实践提供理论与实验依据。方法：1．选用变形链球菌在体外构建符合人体生理环境的人工龋模型，并对人工龋模型中口腔生物膜生长曲线进行测定和釉质块表面显微硬度进行测试。2．PDT防龋剂量的选择：应用不同能量剂量635nm的半导体激光和血卟啉单甲醚(HMME)作用于人工龋，观察PDT过程中不同能量剂量对抑菌效果的影响。3．通过HMME-PDT对细菌生长的抑制、对细菌产酸的抑制，观察PDT对细菌代谢的影响。4．通过显微硬度分析和原子吸收光谱分析PDT对牙齿表面硬度和钙含量的影响。5．通过荧光显微镜结合DiO、Hoechest33342两种荧光探针，探讨PDT在细胞水平的防龋机制。结果：1．口腔细菌生物膜在48 h生长达到了成熟期。致龋处理后，人工龋组牙釉质表面的显微硬度值(MHV)显著低于对照组(P＜0．05)。2．当半导体激光能量剂量为12．47 J／cm~2时，人工龋模型中变形链球菌的生长受到显著抑制(P＜0．05)。当光敏剂HMME剂量适中(20μg／m1)时，PDT才能有效发挥对人工龋口腔生物膜中变形链球菌的杀菌效应(P＜0．05)。3．PDT防龋处理后，人工龋口腔生物膜中变形链球菌数量(CFU／ml)显著减少(P＜0．05)，杀菌率高达99．36％；变形链球菌产酸能力显著下降(P＜0．05)。4．PDT防龋处理后各实验组MHV由大到小顺序为：单纯激光照射＞PDT防龋处理＞单纯HMME＞0．05％洗必泰＞0．9％生理盐水；各实验组钙溶出浓度由小到大顺序为：单纯激光照射＜PDT防龋处理＜0．05％洗必泰＜单纯HMME＜0．9％生理盐水。各实验组间比较具有显著性差异(P＜0．05)。5．在HMME-PDT防龋过程中，HMME可选择性与致龋变形链球菌结合，菌体表面呈现红色荧光；同一视野下，当加入细胞膜绿色荧光探针DiO时，菌体表面呈现绿色荧光；当同时还加入Hoechest33342时，虽然基底可见兰色荧光，但未见Hoechest33342与变形链球菌结合。结论：．采用本研究方法形成的人工龋模型已具备牙釉质早期龋的表现，可用于PDT防龋的实验研究。通过实验选定的HMME-PDT防龋剂量为：HMME剂量为201μg／ml，半导体激光照光剂量为10mW，90s，12．74J／cm~2。PDT可有效杀灭人工龋口腔生物膜中的变形链球菌，并抑制变形链球菌的产酸能力。PDT防龋可减少牙齿钙溶出量，进而显著提高牙齿表面硬度，有利于减慢或阻止龋病的进程。HMME-PDT在防龋过程中的作用靶点位于变形链球菌细胞膜上，激光照射时产生的单态氧和其它活性氧物质(ROS)直接损伤细菌细胞膜系统，影响其新陈代谢，导致细菌死亡，从而取得防龋效果。HMME-PDT是一种有效的防龋方法，在龋病预防中具有广泛的应用前景。更多还原

【Abstract】 Dental caries is harmful to human health of the third kind of disease,secondonly to its health risks of cardiovascular disease and cancer.The direct cause of dentalcaries is that cariogenic Streptococcus mutans and other microorganism formeddental plaque in oral biofilm and acid metabolism activity carried out.Therefore,itcan prevent dental caries for adoption of appropriate means to affect the oral bacteriabiofilm metabolism and interfere with dental plaque formation or the promotion ofdissociation of dental plaque.Although it has been universally recognized of fluoridein caries prevention,but the use of the safety of fluoride and fluoride-resistant strainsof Streptococcus mutans bring a new topic for caries prevention.It is an urgent needof solve the problem to looking for a more safe and effective method of cariesprevention.Photodynamic therapy(PDT) has become one of the basic treatment of cancer.Inrecent years,PDT has also achieved remarkable effects of non-tumor diseaseparticularly for the treatment of bacterial and viral diseases.In view of the foreignlaboratory have been carried out the experimental studies for PDT on a singlecariogenic bacteria,but not on artificial caries model,this study from the point ofview of caries prevention to explore the effect of PDT to cariogenic Streptococcusmutans of oral biofilm of artificial caries model.Objective:To build an artificial caries model consistent with the physiological environmentand used it in PDT anti- caries experiments.To discuss the effects of PDT on themainly oral cariogenic biofilm bacteria (Streptococcus mutans).To analysis theimpact of different doses of PDT photosensitizer to bacteriostasis and select the doseof laser energy for PDT anti- caries.To investigate the anti- caries mechanism of PDTcombined with DiO、Hoechest33342 two fluorescent probe at cell level.To provideacademic and experimental evidence on PDT anti- caries for clinical practice.Methods:1.An artificial caries model was built consistent with the physiological environment with Streptococcus mutans.The growth curve of oral cariogenic biofilm and thesurface micro-hardness of enamel blocks were determined subsquently.2.Selected doses of PDT anti- caries:Applying different doses of 635nm diode laserenergy and different doses photosensitizer to achieve bacteriostasis effects forPDT anti- caries.3.Observed the effects of PDT to bacterial metabolism through the experiments ofinhibition of bacterial growth and acid metabolism of PDT.4.Analyzed the impact of PDT on the tooth surface hardness and calcium content bymicro-hardness analysis and atomic absorption spectrometry.5.Investigated the anti- caries mechanism of PDT combined with DiO、Hoechest33342 two fluorescent probe at cell level.Results:1.Oral bacterial biofilm growth at 48h reached a mature stage.After cariogenictreatment,the enamel surface micro-hardness value(MHV) of artificial cariesgroup was significantly lower than that of the control group(P＜0.05).2.When the diode laser energy dose of 12.47 J/cm~2,the growth of Streptococcusmutans of artificial caries model were significantly inhibited (P＜0.05).When thephotosensitizer HMME moderate dose (20μg/ml),PDT can be effectively playedthe bactericidal effect to Streptococcus mutans of oral biofilm in artificial cariesmodel (P＜0.05).3.After PDT treatment against dental caries,the number of Streptococcus mutans(CFU/ml) of oral biofilm in artificial caries model significantly reduced(P＜0.05),the bactericidal rate up to 99.36%;the ability of S.mutans acidproduction decreased significantly (P＜0.05).4.MHV descending order of the experimental groups:simple laser＞PDT anti-cariestreatment＞simple HMME＞0.05% chlorhexidine＞0.9% saline.The dissolution ofcalcium concentration from small to big as following order:simple laser＜PDTanti-caries treatment＜0.05% chlorhexidine＜simple HMME＜0.9% saline.Therewere significant different compared between the experimental groups (P＜0.05).5.During the process of HMME-PDT anti-caries,HMME optional combined withcariogenic S.mutans showing red fluorescent;the same field of vision,when added green fluorescent membrane probe DiO,the cell surface displayed greenfluorescent;when at the same time also added Hoechest33342,while bluefluorescence in the bacement could be seen,but no Hoechest33342 combinationwith S.mutans.Conclusions:The artificial caries model formed in this study had the performance of earlyeanemal caries and could be used for the experimental research of PDT anti-caries.HMME-PDT doses selected through the experiments were:HMME 20μg/ml,diodelaser 10mW,90s,12.74J/cm~2.PDT can effectively kill S.mutans of artificial cariesoral biofilm and inhibit acid production of S.mutans.PDT can reduce the amount ofcalcium dissolution of the teeth,thereby significantly improve the surface hardness ofthe teeth Will help slow down or prevent the process of dental caries.The target ofHMME-PDT anti-caries located on the membrane of the S.mutans cells.Singletoxygen and other reactive oxygen substances(ROS) produced after laser irradiationdirectly damage bacterial cell membrane system,affecting their metabolism,leading tobacterial death,thereby made anti-caries effect.HMME-PDT is an effective method indental caries prevention,it has broad application prospects in dentistry.更多还原