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浙江大学学报(医学版)  2017, Vol. 46 Issue (2): 118-126    DOI: 10.3785/j.issn.1008-9292.2017.04.02
专题报道     
细菌外膜囊泡纳米载体的制备及其免疫调节作用
陈琪(),吴敏,白宏震,郭则灵,周峻,王青青,汤谷平*()
浙江大学化学系, 浙江 杭州 310028
Bacterial outer membrane vesicles as nano carriers to study immunological activities
CHEN Qi(),WU Min,Bai Hongzhen,Guo Zeling,ZHOU Jun,WANG Qingqing,TANG Guping*()
Department of Chemistry, Zhejiang University, Hangzhou 310028, China
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摘要:

目的:以细菌外膜囊泡(OMV)结合三嵌段聚合物普朗尼克F127(PEO100-PPO65-PEO100),构建能够有效促进巨噬细胞分泌抗肿瘤细胞因子的纳米载药体系OMV-F127。方法:培养减毒沙门菌,用超滤离心法与超声破碎法提取OMV,机械挤压法制得OMV-F127;蛋白质定量试剂盒和SDS-PAGE法检测OMV的蛋白含量和成分;荧光显微镜、透射电镜、动态光学散射法检测OMV-F127形态学特征、粒径、电位及其稳定性;以小鼠巨噬细胞RAW246.7为细胞模型,ELISA检测OMV-F127对抗肿瘤细胞因子的分泌作用。结果:两种方法成功提取到减毒沙门菌的OMV,其蛋白质总量分别为2.8 mg/mL和2.7 mg/mL。成功制备OMV-F127且含有OMV的标志蛋白OmpF/C。OMV为纳米级囊泡结构,F127以及OMV-F127为球形纳米颗粒。OMV、F127、OMV-F127颗粒平均直径分别为(72±2)nm、(90±3)nm、(92±2)nm。OMV-F127能促进抗肿瘤细胞因子IF-12、α-TNF、γ干扰素的分泌。结论:基于细菌OMV的纳米载体OMV-F127能够有效促进巨噬细胞分泌抗肿瘤细胞因子,具有免疫调节作用。

关键词: 沙门氏菌菌苗佐剂,免疫细菌外膜蛋白质类纳米结构聚合物/药理学巨噬细胞细胞因子类细胞,培养的    
Abstract:

Objective: To prepare a nano-carrier based on combining bacterial outer membrane vesicles (OMV) with three block polymer pluronic F127 (PEO100-PPO65-PEO100) (OMV-F127) and to investigate its immunological activity. Methods: Attenuated salmonella (sal) was cultivated. OMV were separated by centrifugal ultrafiltration or ultrasonication, and OMV-F127 was prepared by mechanical extrudation method. The protein contents and compositions were tested with BCA and SDS-PAGE; the morphology of OMV, F127 and OMV-F127 were observed with FM and TEM; the particle sizes and their zeta potential were determined with DLS. Mouse macrophage RAW246.7 cells were treated with OMV-F127 (50 μg/mL, 100 μg/mL) in vitro, and the concentrations of IL-12, TNF-α and IFN-γ in culture supernatant were measured with ELISA kits. Results: The contents of protein in separated OMV by centrifugal ultrafiltration and ultrasonication were 2.8 mg/mL and 2.7 mg/mL, respectively. SDS-PAGE showed the marker protein OmpF/C in OMV. Under the FM and TEM, ball-like structure of F127 and OMV-F127 was observed. Size analysis revealed that the diameters of OMV, F127 and OMV-F127 were 72±2 nm, 90±3 nm and 92±2 nm, respectively. ELISA tests revealed that OMV-F127 significantly stimulated the secretion of IL-12, TNF-α and IFN-γ in RAW246.7 cells. Conclusion: A nano-carrier based on bacterial outer membrane vesicles has been prepared, which can stimulate the secretion of cytokines and may have immunomodulatory effects.

Key words: Salmonella vaccines    Adjuvants, immunologic    Bacterial outer membrane proteins    Nanostructures    Polymers/pharmacology    Macrophages    Cytokines    Cells, cultured
收稿日期: 2016-11-02 出版日期: 2017-10-31
基金资助: 国家自然科学基金(21374098)
通讯作者: 汤谷平     E-mail: cq-chem@zju.edu.cn;tangguping@zju.edu.cn
作者简介: 陈琪(1993—),女,博士研究生,主要从事生物医用纳米材料研究;E-mail: cq-chem@zju.edu.cn;http://orcid.org/0000-0002-5152-0079
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引用本文:

陈琪,吴敏,白宏震,郭则灵,周峻,王青青,汤谷平. 细菌外膜囊泡纳米载体的制备及其免疫调节作用[J]. 浙江大学学报(医学版), 2017, 46(2): 118-126.

CHEN Qi,WU Min,Bai Hongzhen,Guo Zeling,ZHOU Jun,WANG Qingqing,TANG Guping. Bacterial outer membrane vesicles as nano carriers to study immunological activities. J Zhejiang Univ (Med Sci), 2017, 46(2): 118-126.

链接本文:

http://www.zjujournals.com/xueshu/med/CN/10.3785/j.issn.1008-9292.2017.04.02        http://www.zjujournals.com/xueshu/med/CN/Y2017/V46/I2/118

图 1  细菌外膜囊泡纳米载体的构建及被巨噬细胞吞噬过程
图 2  减毒沙门菌8 h内的生长曲线
图 3  BCA测定蛋白标样的标准曲线
图 4  SDS-PAGE检测结果
图 5  OMV在荧光显微镜下表现
图 6  OMV-F127在透射电镜下表现
($\bar x \pm s$)
种类 粒径(nm) 电位(mV)
 OMV 72.3±1.8 -40.9±0.9
 F127 89.2±3.1 -5.3±0.7
 OMV-F127 92.0±2.1 -16.8±3.2
表 1  OMV、F127、OMV-F127的粒径、电位一览
图 7  OMV与OMV-F127的稳定性比较
图 8  OMV-F127对小鼠巨噬细胞RAW246.7分泌γ干扰素、α-TNF、IL-12的作用
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