APDT影响着色芽生菌病病原菌毒力及宿主局部免疫的研究

Chromoblastomycosis (CBM) is a chronic infection caused by dematiaceous fungi which affect the skin and subcutaneous tissue, and is characterized by granulomatous immune response.Fonsecaea monophora is the main causative agents of chromoblastomycosis in southern China.The low cure and high relapse rates are mainly associated with the formation of sclerotic bodies, which can resistant to antifungal drug. The cell divisions cycle 42 (Cdc42) gene has been characterized in the fungi, which plays important roles during growth and development. Quantitative real time RT-PCR revealed that the partial cDNA sequence of Cdc42 of F.monophora, designated FmCdc42 was the highest expression in the sclerotic bodies' stage compared with that in the mycelia and conidia stages, which indicated that the FmCdc42 may be involved in formation of F. monophora sclerotic bodies. System application of antifungal drug takes long time and has side effects. antifungal photodynamic therapy (APDT) is a selective, non-invasive modality of treatment with non-toxic side effects and it will become an effective supplementary mean for the infection which is refractory to conventional treatments. We will use colony forming unit phototoxicity assay to search effective photosenseitisers on sclerotic bodies, then detect the effect of APDT by XTT metabolic assays and the mechanism of APDT by quantitative real-time PCR as well as westernblot for the expression of Cdc42. At last the effective photosensitisers will be used on the animal mode to detect the change of local immune state. The purpose of this project is to ascertain the role and mechanism of APDT on sclerotic bodies, especially on the Langerhans cells. This study will provide a reliable scentific basis for clinical application.

着色芽生菌病是由暗色真菌引起的皮肤及皮下组织感染，广东地区多发，病原菌具有其独特性，以F.monophora多见。该病顽固易复发，主要与病原菌在体内形成硬壳小体抵抗药物杀伤有关。我们已证实参与调节细胞形态变化的细胞分裂周期蛋白42在硬壳小体中的表达量明显高于菌丝和分生孢子，可能参与硬壳小体形成。系统应用抗真菌药疗程长、副作用大；光动力疗法为物理疗法，以选择性破坏靶组织而不累积毒性而成为治疗顽固感染的有效辅助手段。该项目旨在应用不同光敏剂及其特定光源对硬壳小体进行光毒性试验，通过检测实施光动力后真菌代谢、生长情况及Cdc42的表达变化，筛选出有效光敏剂及光源；进一步将其应用于成功构建的着色芽生菌病动物模型上，采用免疫组化观察局部组织中朗格汉斯细胞的聚集，并检测其抗原提呈能力与所分泌的细胞因子水平以判断光动力对局部免疫状态的改变，从而探明该疗法应用于治疗着色芽生菌病的作用靶点，为临床提供依据。

着色芽生菌病，是由暗色孢科中的一组致病性暗色真菌侵犯皮肤和皮下组织引起的慢性疾病。该病在世界各地均有报道，以热带、亚热带地 区多见，近年发病率在全世界呈上升趋势。在我国南方病原菌则主要为 Fonsecaea monophora，且其病原特征导致治疗极其困难。抗真菌的光动力学治疗（APDT）近年来作为辅助治疗应用于着色芽生菌病有肯定疗效，但其作用机制仍不明确。本研究从病原菌及宿主两方面证实不同光敏剂对F. monophora均有一定杀伤作用，其中卟啉类光敏剂杀伤效果显著。各类光敏剂定向累积在病原体内，在光照下通过产生活性氧（ROS）对病原菌进行杀伤。研究结果还表明APDT作用的F. monophora孢子能减低F. monophora感染后巨噬细胞的凋亡，同时降低巨噬细胞抑制率，但其对巨噬细胞膜电位及增殖率的间接调控机制尚不清楚，可能涉及多种复杂的活性信号分子及生物化学信号通路。该研究为APDT临床治疗着色芽生菌病提供理论依据。