两种提取铜绿假单胞菌外膜囊泡的方法比较  

Comparison of two methods of extraction outer membrane vesicles from Psedomonas aeruginosa

作　　者：张佳星[1] 沈犁[1] 华子瑜[2] 刘岚[1] 周薇[1] 姜俊如 

机构地区：[1]重庆医科大学附属儿童医院重庆市儿童发育重大疾病诊治与预防国际科技合作基地儿科学重庆市重点实验室儿童发育疾病研究省部共建教育部重点实验室,重庆400014 [2]重庆医科大学附属儿童医院新生儿科,重庆400014

出　　处：《上海交通大学学报（医学版）》2014年第3期279-282,共4页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81071417/H2002)~~

摘　　要：目的对两种方法提取的铜绿假单胞菌(PA)的外膜囊泡(OMV)的纯度、产量及外毒素A(ETA)含量进行比较。方法分别采取超滤浓缩法和Optiprep密度梯度离心法提取PA的OMV,负染电镜检测形态及纯度;BCA法检测两种方法提取的OMV产量并用Western blotting对OMV中所含ETA进行比较;建立OMV感染人肺癌上皮细胞A549模型并检测IL-8表达水平。结果超滤浓缩法提取的OMV中存在明显的细胞碎片、鞭毛等杂质,产量为21.3μg/1010个细菌,密度梯度离心提取的OMV无明显杂质存在,产量为48.7μg/1010个细菌;相比于超滤浓缩法,密度梯度离心法提取的OMV中含有更高浓度的ETA且诱导A549细胞产生更高水平的IL-8。结论 Optiripe密度梯度离心法提取的PA的OMV的产量和纯度和ETA含量以及激活宿主细胞炎性应答的能量均高于超滤浓缩法。Objective To compare two methods for extracting the outer membrane vesicle （OMV） from Pseudomonas aeruginosa （PA） and analyze it＇s purity, yield, and the content of exotoxin A （ETA）. Methods The OMV of PA was extracted by ultrafiltration concentration and Optiprep density gradient centrifugation. The purity, yield, and content of ETA were detected by negative-stain electron microscopy, BCA method, and Western blotting, respectively. Human lung epithelial cells of A549 were immunized with these two OMVs and the IL-8 was determined by ELISA. Results There were more impurities in OMV extracted by ultrafiltration concentration than OMV extracted by Optiprep density gradient centrifugation. The yield of these two OMVs were 21.3 μg/10^10 bacteria and 48. 7μg/10^10TM bacteria respectively. OMV extracted by Optiprep density gradient centrifugation had more ETA and stronger ability to increase the IL-8 level of A549 cell. Conclusion The OMV of PA extracted by Optiprep density gradient centrifugation have higher purity, yield, and content of ETA, and can induce stronger inflammatory response of host cells.

关 键 词：铜绿假单胞菌 外膜囊泡 超滤浓缩 密度梯度离心 外毒素A IL-8 

分 类 号：R378.991[医药卫生—病原生物学]