5-氨基酮戊酸光动力疗法体外抑制断发毛癣菌肉芽肿株增殖研究

In vitrostudy of the 5-aminolevulinic acid photodynamic therapy for Trichophyton tonsurans strain isolated from granuloma

目的探讨5-氨基酮戊酸光动力疗法(5-Aminolevulinic acid photodynamic therapy,ALA-PDT)对体外培养的断发毛癣菌肉芽肿株的杀伤效应。方法将临床Majocchi肉芽肿患者皮损分离培养出的断发毛癣菌肉芽肿株,在马铃薯葡萄糖琼脂培养基上活化后制备菌悬液,与5mmol/L ALA共孵育后荧光显微镜观察原卟啉PpⅨ(ProtoporphyrinⅨ,PpⅨ)产生情况。实验分对照组、单药组、单光组和ALA-PDT组。对照组无处理。ALA-PDT组断发毛癣菌与5 mmol/L ALA共孵育6h后,采用功率密度为40mW/cm2的633nm红光以不同能量密度(50、100、150、175、200J/cm2)照射。单药组断发毛癣菌与5mmol/L ALA共孵育6h,不照光。单光组无ALA共孵育,只给予200J/cm2红光照射。通过数码相机拍摄照片及菌浓度评估ALA-PDT对断发毛癣菌肉芽肿株生长的抑制效应。结果荧光显微镜下可见明显砖红色荧光物质PpⅨ产生,当照光能量密度为175、200J/cm2时,照片显示菌落生长明显受限,数量减少,菌浓度计数分别为(0.53±0.058)×105 CFU/mL、(0.13±0.057)×105 CFU/mL,明显低于对照组、单药组、单光组及其他低能量密度ALA-PDT组(P<0.05)。结论 ALA-PDT对断发毛癣菌肉芽肿株有明确的杀伤效应,可为临床ALA-PDT治疗皮肤癣菌感染提供理论依据。

Objective To demonstrate the fungicidal effect and action mechanism of 5-aminolevulinic acid photodynamic therapy（ALA-PDT）against Trichophyton tonsurans strain isolated from granuloma.Methods The clinical isolate of Trichophyton tonsurans granuloma was cultured in potato dextrose agar medium to prepare the suspension.After 6 hours of incubation,the production of PpⅨ was observed by fluorescence microscopy.Following PDT（at light energy density of 50,100,150,175,or 200 J/cm^2）,the growth inhibitory of this T.tonsurans strain was observed by colony cultivation.Results Fluorescence microscopy showed obvious PpⅨ production of brick red fluorescent substance.At the light energy density of175 and 200 J/cm^2,the significantly restricted growth of colonies was evidenced by decreased concentration of suspension.The suspension concentrations were（0.53±0.058）×10^5 CFU/mL and（0.13±0.057）×10^5 CFU/mL at light energy density of 175 and 200 J/cm^2 respectively,which were significantly lower than those of control group,single drug group,single light group and other low energy density ALA-PDT group（P〈0.05）.Conclusion ALA-PDT exerted fungicidal effect against Trichophyton tonsurans granuloma isolate,which might provide a theoretical basis for the clinical treatment of dermatophytes.