基于超滤浓缩法和差速离心法制备细菌OMVs的方法探讨  

Comparison between ultrafiltration and differential centrifugation method for concentration of bacterial OMVs

作　　者：陈光璋 范芳芳 梁坤鹏[2] 韦莉[1,3] CHEN Guang-zhang(Anhui Provincial Key Laboratory of Infection and Immunity,Bengbu Medical College,Bengbu 233030,China)

机构地区：[1]蚌埠医学院感染与免疫安徽省重点实验室 [2]蚌埠医学院第二附属医院检验科 [3]蚌埠医学院病原生物学教研室,安徽蚌埠233030

出　　处：《牡丹江医学院学报》2022年第6期9-13,共5页Journal of Mudanjiang Medical University

基　　金：安徽省高校自然科学研究项目(KJ2021A0722);安徽省高校优秀青年人才支持计划项目(gxyq2018037);蚌埠医学院2021年度研究生科研创新计划项目(Byycxz21011)。

摘　　要：目的比较超滤浓缩法、差速离心法提取细菌外膜囊泡(OMVs)的效率,并观察两种方法提取的OMVs对细胞存活率影响的差异。方法分别采取超滤浓缩法和差速离心法提取大肠埃希菌(E.coli)的OMVs,BCA法检测两种方法提取的OMVs蛋白浓度;负染电镜检测其形态及纯度;纳米颗粒跟踪分析检测OMVs粒径大小分布和浓度;CCK8法检测OMVs对细胞存活率的影响。结果两种方法提取的OMVs均为形态典型的球状囊泡结构,差速离心法提取的OMVs的存在包膜不完整,有缺口的情况,产量为(2.7±0.1)μg/10^(10)CFU;超滤浓缩法提取OMVs的产量为(3.3±0.3)μg/10^(10)CFU,高于差速离心法(P<0.05);差速离心法提取的OMVs直径<247.5 nm囊泡数量占总量(82.3±4.6)%,超滤浓缩法提取的OMVs平均粒径更加集中,直径<247.5 nm的囊泡数量占总量(88.8±3.7)%,占比更高(P<0.05);CCK8法检测发现两种方法提取的OMVs均对RAW264.7细胞存活率产生影响(P均<0.05),差速离心法和超滤浓缩法提取的OMVs作用后,细胞存活率分别为(84.0±4.6)%和(74.1±6.6)%,组间无显著性差异(P>0.05)。结论超滤浓缩法提取的E.coli的OMVs的完整性、纯度和产量均高于差速离心法,具有更高的提取效率,两种方法提取的OMVs对细胞存活率的影响无差异。Objective To compare the extraction efficiency of bacterial outer membrane vesicles(OMVs)between ultrafiltration concentration method and differential centrifugation method,and to observe the effect on cell survival rate.Methods The OMVs of E.coli was extracted by ultrafiltration concentration method and differential centrifugation method,respectively.Protein concentration of samples was quantified by BCA method.The morphology and purity of OMVs were detected by negative staining electron microscope,and the particle size distribution and concentration were detected by nano particle tracking analysis(NTA).The survival rate of cells was detected by CCK-8assay.Results The OMVs extracted by the two methods were typical spherical vesicles.Compared with ultrafiltration concentration method,OMVs extracted by differential centrifugation method had incomplete envelope and the protein concentration of OMVs was(2.7±0.1)μg/10^(10)bacterial cells.and the protein concentration of OMVs extracted by ultrafiltrationcon centration method was(3.3±0.3)μg/10^(10)bacterial cells.The yield of OMVs extracted by ultrafiltration concentration method was higher than that by differential centrifugation(P<0.05).Then umber of vesicles with OMV sless than 247.5 nm extracted by differential centrifugation accounted for(82.34.6)%,and the number of vesicles with OMVs less than 247.5 nm extracted by ultrafiltration concentration accounted for(88.83.7)%.Compared with differential centrifugation,the average particle size of OMVs extracted by ultrafiltration concentration method was more concentrated,and the proportion of vesicles less than 250 nm was higher(P<0.05).The results of CCK8 method show that the OMVs extracted by the two methods both affected the survival rate of cells(P<0.05).The survival rate of RAW264.7 cells stimulated by OMVs extracted by differential centrifugation method accounted for(84.04.6)%,and(74.16.6)%by OMVs extracted by ultrafiltration concentration method.But,there was no significant difference between the two groups(P>

关 键 词：大肠埃希菌 外膜囊泡 超滤浓缩 差速离心 

分 类 号：R378.991[医药卫生—病原生物学]