细菌外膜囊泡纳米载体的制备及其免疫调节作用

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。
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