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浙江大学学报（医学版）
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    或    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
1 HU Q , WU M , FANG C et al. Engineering nanoparticle-coated bacteria as oral DNA vaccines for cancer immunotherapy[J]. Nano Lett, 2015, 15 (4): 2732- 2739
doi: 10.1021/acs.nanolett.5b00570
2 COUSSENS L M , WERB Z . Inflammation and cancer[J]. Nature, 2002, 420 (6917): 860- 867
doi: 10.1038/nature01322
3 MERCADO-LUBO R , ZHANG Y , ZHAO L et al. A Salmonella nanoparticle mimic overcomes multidrug resistance in tumours[J]. Nature Commun, 2016, 7 12225-
doi: 10.1038/ncomms12225
4 ZHOU X , ZHANG X , HAN S et al. Yeast microcapsule-mediated targeted delivery of diverse nanoparticles for imaging and therapy via the oral route[J]. Nano Lett, 2017, 17 (2): 1056- 1064
doi: 10.1021/acs.nanolett.6b04523
5 GAO W , FANG R H , THAMPHIWATANA S et al. Modulating antibacterial immunity via bacterial membrane-coated nanoparticles[J]. Nano Lett, 2015, 15 (2): 403- 1409
6 FISSEHA M , CHEN P , BRANDT B et al. Characterization of native outer membrane vesicles from lpxL mutant strains of Neisseria meningitidis for use in parenteral vaccination[J]. Infect Immun, 2005, 73 (7): 4070- 4080
doi: 10.1128/IAI.73.7.4070-4080.2005
7 KUIPERS K , DALEKE-SCHERMERHORN M H , JONG W S et al. Salmonella outer membrane vesicles displaying high densities of pneumococcal antigen at the surface offer protection against colonization[J]. Vaccine, 2015, 33 (17): 2022- 2029
doi: 10.1016/j.vaccine.2015.03.010
8 EWARDS M F , STOCKER B A . Construction of delta aroA his delta pur strains of Salmonella typhi[J]. J Bacteriol, 1988, 170 (9): 3991- 3995
doi: 10.1128/jb.170.9.3991-3995.1988
9 LEE E Y , BANG J Y , PARK G W et al. Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli[J]. Proteomics, 2007, 7 (17): 3143- 3153
doi: 10.1002/(ISSN)1615-9861
10 ROY N , BARMAN S , GHOSH A et al. Immunogenicity and protective efficacy of Vibrio cholerae outer membrane vesicles in rabbit model[J]. FEMS Immunol Med Microbiol, 2010, 60 (1): 18- 27
doi: 10.1111/fim.2010.60.issue-1
11 李 晓晶 , 高 丹丹 , 毕 研伟 et al. 三种方法提取的B群脑膜炎奈瑟菌外膜囊泡组分的免疫原性、毒性及杀菌活性[J]. 中国生物制品学杂志, 2011, 24 (10): 1183- 1186
LI Xiaojing , GAO Dandan , BI Yanwei et al. Immunogenicity and toxicity of outer membrane vesicle of serogroup b neisseria meningitides extracted by three methods[J]. Chinese Journal of Biologicals, 2011, 24 (10): 1183- 1186
12 ORMAZáBAL M , BARTEL E , GAILLARD ME et al. Characterization of the key antigenic components of pertussis vaccine based on outer membrane vesicles[J]. Vaccine, 2014, 32 (46): 6084- 6090
doi: 10.1016/j.vaccine.2014.08.084
13 ZHANG J , GAO W , FANG R H et al. Synthesis of nanogels via cell membrane-templated polymerization[J]. Small, 2015, 11 (34): 4309- 4313
doi: 10.1002/smll.v11.34
14 GUJRATI V , KIM S , KIM S H et al. Bioengineered bacterial outer membrane vesicles as cell-specific drug-delivery vehicles for cancer therapy[J]. ACS Nano, 2014, 8 (2): 1525- 1537
doi: 10.1021/nn405724x
15 BEATTY G L , PATERSON Y . Regulation of tumor growth by IFN-gamma in cancer immunotherapy[J]. Immunol Res, 2001, 24 (2): 201- 210
doi: 10.1385/IR:24:2
16 TRINCHIERI G , PFLANZ S , KASTELEIN R A . The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses[J]. Immunity, 2003, 19 (5): 641- 644
doi: 10.1016/S1074-7613(03)00296-6
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