上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

两种提取铜绿假单胞菌外膜囊泡的方法比较

张佳星1, 沈 犁1，华子瑜2, 刘 岚1，周 薇1，姜俊如1   

  1. 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
  • 出版日期:2014-03-28 发布日期:2014-04-02
  • 通讯作者: 沈 犁, 电子信箱: shenli80@126.com; 华子瑜, 电子信箱: h-ziyu@163.com。
  • 作者简介:张佳星（1988—）， 女， 硕士生; 电子信箱: zhangjiaxing415@126.com。
  • 基金资助:

    国家自然科学基金（81071417/H2002）

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

ZHANG Jia-xing1, SHEN Li1, HUA Zi-yu2, LIU Lan1, ZHOU Wei1, JIANG Jun-ru1   

  1. 1.Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics of Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children's Hospital of Chongqing Medical University, Chongqing 400014, China; 2.Department of Neonatology, Children's Hospital, Chongqing Medical University, Chongqing 400014, China
  • Online:2014-03-28 Published:2014-04-02
  • Supported by:

    National Natural Science Foundation of China, 81071417/H2002

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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21.3 μg/1010个细菌，密度梯度离心提取的OMV无明显杂质存在，产量为48.7 μg/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。

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

Abstract:

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/1010 bacteria and 48.7 μg/1010 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.

Key words: Pseudomonas aeruginosa, out membrane vesicle, ultrafiltration concentration, density gradient centrifugation, exotoxin A, IL-8