光动力疗法单用或联合抗真菌药物对游离态及形成生物膜的皮炎外瓶霉凋亡的影响

Effects of photodynamic therapy alone or in combination with antifungal agents on the apoptosis of planktonic and biofilm cells of Exophiala dermatitidis

目的 探讨光动力疗法单用或联合抗真菌药物对游离态及形成生物膜的皮炎外瓶霉凋亡的作用。方法 制备皮炎外瓶霉游离态孢子悬液，用改良的96孔板法构建皮炎外瓶霉生物膜。将皮炎外瓶霉游离态孢子和生物膜分别分为单药组（只用抗真菌药物处理）、光动力组（只用光动力处理）和光动力联合药物组（先用光动力处理，再用抗真菌药物处理），同时设立空白对照组（既不用抗真菌药物处理也不用光动力处理），抗真菌药物包括两性霉素B、泊沙康唑、伏立康唑和伊曲康唑，药物浓度均为1 mg/L，处理时间均为2 h。采用末端脱氧核苷酸转移酶介导的dUTP 缺口末端标记（TUNEL）法进行凋亡检测。结果 抗真菌药及光动力疗法对游离态（均P 〈 0.001）及形成生物膜（均P 〈 0.05）的皮炎外瓶霉凋亡率均有影响。空白对照组和两性霉素B、泊沙康唑、伏立康唑、伊曲康唑单药组游离态皮炎外瓶霉凋亡率分别为11.67% ± 0.21%、13.30% ± 1.78%、14.30% ± 3.61%、14.51% ± 1.91%、36.17% ± 4.00%，其中伊曲康唑单药组凋亡率显著高于空白对照组（P 〈 0.05），其他3个单药组与空白对照组差异均无统计学意义（均P 〉 0.05）。光动力组游离态孢子凋亡率（41.37% ± 7.80%）显著高于空白对照组（P 〈 0.05）。光动力联合两性霉素B、泊沙康唑、伏立康唑、伊曲康唑组的游离态孢子凋亡率分别为29.23% ± 6.71%、37.23% ± 10.86%、43.57% ± 6.42%、69.87% ± 3.53%），其中联合伏立康唑、伊曲康唑组凋亡率显著高于相应单药组（均P 〈 0.05）。光动力组生物膜凋亡率（32.00% ± 0.43%）显著高于空白对照组（25.30% ± 1.31%，P 〈 0.05），光动力联合两性霉素B组显著高于两性霉素B组（P 〈 0.05）。结论 光动力联合抗真菌药物能够明显促进游离态及形成生物膜的皮炎外瓶霉的凋亡。

Objective To evaluate the in vitro effects of photodynamic therapy alone or in combination with antifungal agents on the apoptosis of planktonic and biofilm cells of Exophiala dermatitidis （E. dermatitidis）. Methods The planktonic suspensions of E. dermatitidis were prepared, and the biofilms of E. dermatitidis were prepared via a modified 96-well plate-based methods. Planktonic and biofilm cells of E. dermatitidis were separately divided into several groups： antifungal agent groups treated with antifungal agents alone, photodynamic therapy group receiving photodynamic therapy alone, combination groups receiving photodynamic therapy followed by the treatment with antifungal agents, and blank control group receiving no treatment. These antifungal agents included amphotericin B, posaconazole, voriconazole and itraconazole. The concentrations of these antifungal agents were all 1 mg/L, and the treatment with antifungal agents lasted 2 hours. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay was performed to detect the apoptosis of planktonic and biofilm cells of E. dermatitidis in all the groups. Results The antifungal agents and photodynamic therapy both affected the apoptosis of planktonic （both P 〈 0.001） and biofilm cells （both P 〈 0.05） of E. dermatitidis. The apoptosis rates of E. dermatitidis planktonic cells in the control group, amphotericin B group, posaconazole group, voriconazole group and itraconazole group were 11.67% ± 0.21%, 13.30% ± 1.78%, 14.30% ± 3.61%, 14.51% ± 1.91% and 36.17% ± 4.00% respectively. The apoptosis rate of E. dermatitidis planktonic cells was significantly higher in the itraconazole group than in the control group（P 〈 0.05）, but no significant differences were observed between the other 3 antifungal agent groups and control group （all P 〉 0.05）. The photodynamic therapy group also showed a significantly higher apoptosis rate of E. dermatitidis planktonic cells （41.37% ± 7.80%） compared with the control group （P 〈 0.05）. After the treatment with photodynamic therapy combined with amphotericin B, posaconazole, voriconazole or itraconazole, the apoptosis rates of E. dermatitidis planktonic cells were 29.23% ± 6.71%, 37.23% ± 10.86%, 43.57% ± 6.42% and 69.87% ± 3.53% respectively. Moreover, the photodynamic therapy ＋ voriconazole group and photodynamic therapy ＋ itraconazole group both showed significantly higher apoptosis rates compared with the voriconazole group and itraconazole group respectively（both P 〈 0.05）. The apoptosis rate of E. dermatitidis biofilm cells was significantly higher in the photodynamic therapy group than in the control group（32.00% ± 0.43% vs. 25.30% ± 1.31%, P 〈 0.05）, as well as in the photodynamic therapy ＋ amphotericin B than in the amphotericin B group （P 〈 0.05）. Conclusion Photodynamic therapy combined with antifungal agents can markedly promote the apoptosis of planktonic and biofilm cells of E. dermatitidis.