新型光敏剂Ru化合物介导的光动力对铜绿假单胞菌的体外杀伤效应  

Antibacterial Activity of Ru Photosensitizer: An in vitro Study on Pseudomonas Aeruginosa Strains

作　　者：吴苏敏[1] 王钰铖[1] 甄洁[1] 王颖[1] 顾瑛[1] 

机构地区：[1]解放军总医院激光医学科,北京市100853

出　　处：《中国激光医学杂志》2014年第1期1-5,56-57,共7页Chinese Journal of Laser Medicine & Surgery

基　　金：国家自然科学基金(81171633);北京市自然科学基金(4132079)

摘　　要：目的观察新型光敏剂Ru化合物介导的光动力（Ru complex mediated Photodynamic Therapy，Ru-PDT）对5株离体铜绿假单胞菌（Pseudomonas aeruginosa）的杀伤作用，并初步研究PDT杀菌的作用位点。方法分离培养两株标准菌株ATCC27853、PA01（即ATCC15692）和3株临床耐药菌株，制备成-10^8CFU／ml的细菌悬液。实验分为4组：PDT组、单纯光敏剂组、单纯照光组和空白对照组。（1）PDT组：不同浓度Ru光敏剂（终浓度为0．1、0．25、1．0、2．5、10和25μM）与细菌悬液混合后避光孵育30min，用457nln激光照射，照射时间10min，功率密度为40mW／cm2。（2）单纯光敏剂组：50μM光敏剂Ru化合物与细菌悬液避光混合，孵育30min，不予激光照射。（3）单纯照光组：同浓度细菌悬液与PBS混合后，采用波长457nln激光照射，照射时间10min，功率密度为40mW/cm2。（4）空白对照组：将细菌悬液与PBS混合后，不加入光敏剂，不予激光照射。将各组处理后的细菌悬液收集，10倍递增连续稀释后涂板培养24h进行菌落计数。将PDT处理前后的细菌收集固定，制备超薄切片，通过透射电镜观察PDT作用后菌体的形态学变化。结果在本实验的光敏剂浓度范围内，PDT对铜绿假单胞菌的杀伤作用呈光敏剂浓度依赖性，以10μM光敏剂Ru，PDT处理5株菌均能达到有效杀菌。单纯照光或光敏剂孵育对细菌存活无影响。电镜观察发现PDT处理组细菌膜结构破坏严重，细菌内容物外溢。结论Bu-PDT能对多重耐药铜绿假单胞菌有很好的体外杀菌作用；临床耐药菌的膜结构是其产生杀菌作用的重要位点。Objective To assess the killing efficacy of Ru complex mediated photedynamic therapy （Ru-PDT） on 5 isolates of Pseudomonas aerug- inosa in vitro and observe the action site of Ru-PDT process. Methods Bacterial suspension of 2 standard strains, namely, ATCC 27853 and PAO1 （ATCC15692） and 3 clinical drug-resistant strains of Pseudomonas acruginosa were prepared to form a concentration of - 10^8 CFU/ml. The bacteria were divided into 4 groups. The PDT group ： photosensitizers of different concentrations （ the final concentrations of PSs were 0. 1, 0. 25, 1.0, 2. 5,10 and 25 μM） and the bacterial suspension were mixed and incubated in dark for 30 min. Illumination of 457 nm laser was then performed for 10 min with a power density of 40 mW/cm2. The photosensitizer group： the PS of 50μM was incubated with bacterial suspension for 30 min, no illumination followed. The light group： the bacterial suspension was exposed to 40 mW/cm2 457 nm laser light in the absence of PS. The blank control group： bacterial suspension was mixed with PBS, without PS or illumination. The bacterial samples were collected respectively after each of the treatments and the survival were calculated with counting colony-forming units （CFUs）. The bacterial samples were fixed before and after the treatment. Ultra-thin sections were prepared and morphological changes were observed using transmission electron microscope （TEM）. Results Neither 457 nm laser nor PS had any effect on the viability of Pseudomonas aemginosa. The killing effect on these five strains increased as the concentration of PS increased. The viability of tested bacteria fell more than 4 log at 10 μM, which represents a value greater than 99. 99% of cellular inactivation. The damage of the bacterial membrane structure and leakage of the cytoplasm was ob- served in the PDT group. Conclusions Ru-PDT can effectively kill Pseudomonas aeruginosa in vitro; membrane structure was an important target of the PDT bactericidal process.

关 键 词：铜绿假单胞菌 光动力疗法 钌化合物 透射电镜 

分 类 号：R446.5[医药卫生—诊断学]