肺炎克雷伯菌外膜囊泡的提取、鉴定及相对定量

Extraction,identification and relative quantification of outer membrane vesicles of Klebsiella pneumoniae

目的基于超速离心改良法快速分离提取肺炎克雷伯菌分泌的外膜囊泡并进行鉴定和定量。方法将购于国家卫生健康委员会临床检验中心经典肺炎克雷伯菌(cKP)标准菌株和台湾大学馈赠高毒力肺炎克雷伯菌(hvKP)标准菌株分别置于M9基础培养基中培养9 h,利用超速离心改良法提取外膜囊泡(OMV),然后采用负染法于透射电子显微镜下观察其形态和大小,通过Stewart磷脂分析法进行相对定量,并采用两独立样本t检验进行两组间比较。结果透射电子显微镜下观察发现,cKP和hvKP分泌OMV的形态大部分呈球形、囊状小泡结构,小部分形态不规则,OMV直径为20~250 nm,可见多个囊泡聚集成团,且hvKP分泌的OMV多于cKP。相对定量检测发现,hvKP分泌的OMV数量多于cKP,差异有统计学意义(P<0.05)。结论通过超速离心改良法成功实现了肺炎克雷伯菌OMV提取、鉴定及定量检测,为后续OMV机制及功能研究奠定了基础,为抗细菌感染治疗提供了新思路。

Objective Based on the modified ultracentrifugation method,the outer membrane vesicles(OMV)secreted by Klebsiella pneumoniae were rapidly separated,identified and quantified.Methods Standard strains of classic Klebsiella Pneumoniae(cKP)purchased from the Clinical Laboratory Center of the National Health Commission,and hypervirulent Klebsiella pneumoniae(hvKP)which was donated by Taiwan University were cultured in M9 basal media for 9 hours,and the OMV were extracted by modified ultracentrifugation.The shape and size of OMV were identified by transmission electron microscopy(TEM),relative quantification by Stewart phospholipids analysis method.Two groups were compared using independent samples t test.Results It was observed under the TEM that most of the OMV secreted by cKP and hvKP showed spherical vesicle structure and a small part were irregular.The diameter of OMV ranged from 20 to 250 nm,multiple vesicles could be seen in clusters.Relative quantification found that the number of OMV secreted by hvKP were more than cKP(P<0.05).Conclusions This study successfully achieved the extraction,identification and quantification of OMV from Klebsiella pneumoniae through the modified ultracentrifugation method,which provided a foundation for further study about the function and mechanism of OMV,and also provided new ideas for the treatment of bacteria.Based on the ultracentrifugation method,the OMV secreted by Klebsiella pneumoniae were rapidly separated and extracted,then identified and quantified.