声动力诱导牙菌斑中牙龈卟啉单胞菌的脂质与DNA氧化的机制研究

Dental plaque is the initiating factor of periodontal disease, based on the overall survival and difficult to remove the bacterial biofilm. The key to the treatment of periodontal disease is to control plaque. Sonodynamic therapy (SDT), based on the effect of ultrasound combined with sonosensitizer, is a forefront therapeutic modality on inhibit bacteria. However, up to date, the mechanism of SDT on bacteria has not been illuminated which may be associated with bacterial oxidative stress. Reactive oxygen species (ROS) is the main production of bacterial oxidation stress. The effect of ROS in inactivated bacteria induced by SDT has not been reported. Our previous studies have found that SDT can significantly restrain the growth of Staphylococcus aureus and Porphromonas gingivalis (Pg). we hypothesized that SDT induced bacterial oxidation stress increase, ROS produced, is the key to kill bacteria. To prove this hypothesis, dental plaque model is established, under the treatment of SDT, laser scanning confocal microscope is applied to observe the changes in the structure of biofilm, 454 pyrosequencing is used to detect the quantity and quality of the plaque samples, and combined with fluorescence probe and chemiluminescence method to evaluate the changes of ROS production in the biofilm. We expect to provide scientific theory foundation and safe treatment method for the clinical treatment of periodontal disease, illuminate the mechanism of SDT on bacteria by observing the effect of ROS on lipids and DNA in Pg of subgingival dental plaques induced by SDT.

牙菌斑是牙周病的始动因子，是以整体生存又难以清除的细菌性生物膜。牙周病治疗成功的关键是控制菌斑。声动力学疗法（SDT）是超声联合声敏剂抑制细菌的前沿方法，对细菌的杀伤机制尚未阐明，多认为与细菌的氧化压力有关。活性氧是细菌氧化压力升高的主要产物，在SDT灭活细菌中的作用迄今国内外尚无报道。本课题组前期研究证实SDT能有效杀灭金黄色葡萄球菌与牙龈卟啉单胞菌（Pg），我们推测SDT引起细菌内氧化压力升高，产生活性氧（ROS），是导致细菌死亡的关键。为证实这一假说，拟建立牙菌斑模型，在SDT的作用下，应用激光扫描共聚焦显微镜观察生物膜结构的改变、并采用454焦磷酸测序检测各菌种及数量的变化，结合荧光探针和化学发光法评估生物膜中ROS产物的改变。通过观察SDT诱导产生的ROS对牙菌斑生物膜中Pg的脂质与DNA的影响，阐明SDT抑制细菌的机制，为SDT治疗牙周病提供科学的理论依据与安全的治疗方法。

牙菌斑是牙周病的始动因子，是以整体生存又难以清除的细菌性生物膜。牙周病治疗成功的关键是控制菌斑。声动力学疗法(sonodynamic therapy, SDT)是超声联合声敏剂抑制细菌的前沿方法，对细菌的杀伤机制尚未阐明，多认为与细菌的氧化压力有关。活性氧是细菌氧化压力升高的主要产物，在SDT灭活细菌中的作用迄今国内外尚无报道。本课题组前期研究证实SDT能有效杀灭金黄色葡萄球菌与牙龈卟啉单胞菌(Porphyromonas gingivalis, Pg)，我们推测SDT引起细菌内氧化压力升高，产生活性氧物质(reactive oxygen species, ROS)，是导致细菌死亡的关键。在此基础上，本课题组进行以下实验研究，建立牙菌斑模型，描述牙菌斑生物膜的特性，在SDT的作用下，证实血卟啉单甲醚(hematoporphyrinmonomethyl ether, HMME)介导的SDT对牙菌斑生物膜与大鼠牙周炎有明显的治疗效果.应用激光扫描共聚焦显微镜观察在SDT组生物膜结构明显被破坏，并且在SDT组细菌存活数量明显下降，同时检测到ROS产物量增多，丙二醛(MDA)含量以及细胞内外ROS和超氧化物歧化酶(SOD)含量增多。观察到SDT诱导产生的ROS对牙菌斑生物膜中Pg的脂质与DNA的影响，阐明SDT抑制细菌的机制，为SDT治疗牙周病提供科学的理论依据与安全的治疗方法。超声强度与HMME作用浓度等实验参数的筛选对于SDT的作用效果产生直接影响，在实验参数的作用下，明确HMME对人牙龈成纤维细胞与SDT对大鼠牙周组织的生物安全性，对于日后临床应用也具有重要的指导意义。