ROS在1064nm激光治疗红色毛癣菌甲真菌病中的作用机制研究

Trichophyton rubrum（T.rubrum）is the most common causative agent of onychomycosis.The previous research show that lasers could inhibit or kill the fungi in vitro and its mechanism is unclear. The data from our clinical study showed that 60% cases of onychomycosis got a great improvement by laser treatment.We selected two T.rubrum isolates for further study. One is T.rubrum Y with good response to laser irradiation.The other one is T.rubrum N with poor response to laser irradiation.Following to the upgrading laser energy, T.rubrum Y had the increasing ROS level and the obviously destructive ultrastructure by SEM and TEM while T.rubrum N with slight change of reactive oxygen species(ROS) and ultrastructure .It indicated that ROS may play an important role on onychomycosis by laser therapy.In the present study, the changes ,before and after laser treatment on T.rubrum Y and T.rubrum N in vitro , will be compared among Hsp、ROS、the related molecules in mitochondrial apoptosis pathways by real-time PCR、Electron Microscope、fluorescent probe labeling、 TUNEL methods.Then, the study will elaborate and validate the important role of ROS mediated apoptosis during laser treatment by additional intervention of Oxidants and reductant agents. Finally, to evaluate the relationships among therapeutic effect,the level of ROS and thermotolerant degree of T.rubrum isoaltes from ill nails in vivo and to provide theory evidence for the laser treatment on onychomycosis.

红色毛癣菌（T.rubrum）是甲真菌病的主要致病菌。研究表明激光可抑制或杀灭T.rubrum，机制不清。我们前期研究中，激光治疗T.rubrum甲真菌病的有效率约60%。进一步体外研究发现：随着激光剂量增加，激光治疗敏感病甲分离的T.rubrum Y的ROS↑，菌体结构破坏加剧，而不敏感病甲分离的T.rubrum N 的ROS无著变，结构破坏轻微。提示ROS可能在激光治疗甲真菌病中有重要作用。本研究拟1.体外应用定量PCR、荧光探针标记、电镜、TUNEL等方法比较T.rubrum Y和T.rubrum N在激光照射前后Hsp 、ROS及线粒体凋亡通路相关因子变化，并通过外源性氧化及还原剂的干预，阐明及验证ROS介导的凋亡在激光治疗甲真菌病中的作用；2.在体评价激光治疗前后病甲的临床疗效、ROS和病甲分离的T.rubrum热耐受性的关系，为合理选择激光治疗甲真菌病的类型及疗程提供理论依据。

甲真菌病常见多发，红色毛癣菌是甲真菌病的主要致病菌。近年，激光应用于治疗甲真菌病，但机制不明。本课题组在体外通过检测不同能量长脉冲Nd:YAG 1064nm激光照射不同表型红色毛癣菌菌落后ROS量的变化，同时进一步检测在加入氧化剂和还原剂的条件下，ROS的变化，结果表明随着激光照射能量的增加，绒毛型红色毛癣菌菌落的ROS含量线性升高，而沟纹型红色毛癣菌菌落的ROS含量无明显变化，表明ROS在激光治疗甲真菌病中具有重要作用。在此基础上，本课题组通过不同表型红色毛癣菌菌落在不同剂量激光照射后其透射、扫描电镜下菌体结构的变化（凋亡和坏死的表现：髓样小体、蛋白凝固体、菌体崩解、坏死）及线粒体膜电位的变化（线粒体膜电位随着激光能量增加而消失）等，进一步表明ROS介导的线粒体凋亡是激光治疗甲真菌病的基础。最后，课题组通过在体评价激光治疗红色毛癣菌感染的病甲治疗前、治疗后的临床疗效、ROS含量变化、病甲分离的红色毛癣菌对热的耐受性、真菌学指标之间的关系，进一步阐述了ROS在激光治疗甲真菌病中的作用，为激光治疗甲真菌病提供理论依据