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目的观察新型光敏剂亚苄基环戊酮化合物P2(cationic benzylidene cyclopentanone photosensitizers)介导的光动力疗法(photodynamic therapy,PDT)对耐甲氧西林金黄色葡萄球菌(methicillin-resistant staphylococcus aureus,MRSA)生物被膜渗透性的影响,及P2-PDT与左氧氟沙星(Levofloxacin,LVFX)联合应用对生物被膜内细菌的杀伤作用。方法 (1)观察P2-PDT对MRSA生物被膜渗透性的影响。首先建立MRSA生物被膜渗透性检测模型。实验分为空白对照组和PDT组,每组6个样本。PDT组采用P2-PDT处理,P2光敏剂浓度10μM,避光孵育30 min后,采用激光照射,激光波长532nm,功率密度40 mW/cm~2、照射时间10 min,能量密度24 J/cm~2;对照组不进行P2-PDT处理。两组标本处理后,加入浓度为10μg/ml LVFX,6 h后采用高压液相色谱仪测定透过细菌生物被膜的LVFX浓度。(2)观察P2-PDT与LVFX联合应用对生物被膜内细菌的杀伤作用。实验分为空白对照组(A组),即不加入LVFX溶液、不进行PDT照射;单纯LVFX组(B组)即只加入LVFX溶液、不进行PDT照射;单纯PDT组(C组),即只进行PDT照射、不加入LVFX溶液;PDT+LVFX联合组(D组),采用P2-PDT处理后即刻,加入浓度为10μg/ml的LVFX溶液。各组样本在处理后继续培养24 h,采用XTT法检测生物被膜内细菌的存活率。结果 (1)P2-PDT对MRSA生物被膜渗透性的改变,两组透过MRSA生物被膜的LVFX浓度分别为:PDT组(2.02±0.2)μg/ml、空白对照组(1.60±0.2)μg/ml,两组比较差异具有非常显著意义(P<0.01)。(2)P2-PDT与LVFX联合应用对生物被膜内细菌的杀伤作用。四组生物被膜内细菌的存活率分别为:A组(100.0±0.0)%、B组(86.49±3.5)%、C组(57.42±0.9)和D组(0.76±0.6)%,其中D组生物被膜内细菌的存活率显著低于B组和C组,三组比较差异具有非常显著意义(P<0.01)。结论 P2-PDT能够增强MRSA生物被膜对抗生素LVFX的渗透性,P2-PDT与LVFX联合应用对杀伤生物被膜内细菌有协同作用。
Objective To observe the effect of photodynamic therapy (PDT) mediated by a novel photosensitizer, benzyl benzyl cyclopentanone photosensitizers (P2P) on methicillin-resistant staphylococcus aureus (MRSA) Membrane permeability, and P2-PDT and levofloxacin (Levofloxacin, LVFX) combination of biofilm bacteria killing effect. Methods (1) To observe the effect of P2-PDT on the permeability of MRSA biofilm. First, MRSA biofilm permeability test model was established. The experiment was divided into blank control group and PDT group, with 6 samples in each group. The PDT group was treated with P2-PDT, the concentration of P2 photosensitizer was 10μM, and incubated for 30 min in the dark. The laser irradiation, laser wavelength 532 nm, power density 40 mW / cm2, irradiation time 10 min, energy density 24 J / 2; control group did not carry out P2-PDT treatment. After two groups of samples were treated, the concentration of LVFX was added at a concentration of 10μg / ml. After 6 hours, the concentration of LVFX in the biofilm of the bacteria was measured by high pressure liquid chromatography. (2) To observe the killing effect of P2-PDT and LVFX on bacteria in biofilm. The experimental group was divided into blank control group (group A), ie without addition of LVFX solution and no PDT irradiation; LVFX group (group B) was only added LVFX solution without PDT irradiation; PDT group (group C) PDT irradiation without adding LVFX solution; PDT + LVFX combined group (D group), immediately after treatment with P2-PDT, the concentration of 10μg / ml of LVFX solution was added. The samples of each group were cultured for 24 h after treatment, and the survival rate of bacteria in biofilm was detected by XTT method. Results (1) The changes of permeability of MRSA biofilms by P2-PDT showed that the LVFX concentrations in MRSA biofilms were 2.02 ± 0.2 μg / ml in PDT group and 1.60 ± 0.2 μg / ml, with significant difference between the two groups (P <0.01). (2) The killing effect of P2-PDT and LVFX on bacteria in biofilm. The survival rate of bacteria in four groups of biofilms was 100.0 ± 0.0% in group A, 86.49 ± 3.5% in group B, and 57.42 ± 0.9 in group C and 0.76 ± 0.6% in group D, The survival rate of bacteria in biofilm was significantly lower than those in groups B and C, with significant differences among the three groups (P <0.01). Conclusion P2-PDT can enhance the permeability of MRSA biofilm to antibiotic LVFX. The combination of P2-PDT and LVFX has a synergistic effect on the bacteria in the biofilm.