| The outer membrane vesicles,or OMVs briefly,is a nanoscale vesicle with lipid bilayer structure secreted from the cell membrane into extracellular space during the growth of bacteria,which contain a variety of biologically active molecules such bacterial cell membrane proteins,lipids,and nucleic acids that transmit signals between cells and has a variety of physiological functions,such as : mediating bacterial-host interactions,presenting bacterial antigens,and regulating host immune system function.With the depending of research on the biological characteristics and functions of OMVs,whether some symbiotic bacteria and probiotics can induce the development of the host immune system through the pathway of secreting OMVs has attracted widespread attention in the academic community.The experiment was carried out to isolate and purity the extracellular vesicles of Bacillus subtilis 168,and the effect of B.subtilis 168 extracellular vesicles(BS-OMVs)on the viability of macrophages was studied.The recombinant B.subtitls 168 of green fluorescent protein was constructed to detect the ability o extracellular vesicles to carry out recombinant protein.The main result are as follows:(1)The differential centrifugation was used to enrich BS-OMVs from the supernatant of B.subtilis 168 culture medium.Then purified BS-OMSs was purified by density gradient centrifugation.The results of scanning electron microscopy showed that the shape and site of BS-OMVs were different,the filed of vision was clear and no impurity,it was round or oval,the average diameter was about 100 nm,the range was within 50-200 nm,which was in line with the requirements of the particle size of the bacterial OMVs particles;the detection results of the nano particle size analyzer showed that the MODE curve lines were smooth and fluent.The other impurities contained in BS-OMVs were less,the purity was good,the peak diameter of the particle was 85 nm,the particle size range was 50-200 nm,which basically coincided with the size value of the fine particle observed by the SEM.The results of protein quantification showed that the protein content of BS-OMVs of 1 x 1010 particles/mL was 5.1 ug/mL.(2)The effects of 6 groups of different concentrations of BS-OMVs(0,1,2,4,8 and 16 ug/mL)on cell survival,intracellular enzyme activity and secretory cytokines(TNF-? and IL-6)and anti-inflammatory cytokines(IL-10 and TGF-?)were studied in vitro with mouse macrophage RAW264.7 cells as an in vitro model.Compared with the control group PBS,there was no significant difference in cell survival rate,extracellular LDH and intracellular SOD activity in OMV1 and OMV2 groups,the activity of intracellular ACP,LDH and iNOS increased significantly(P<0.05),and the secretion of proinflammatory cytokines(TNF-?and IL-6)increased to 2-5 and 3-5 times that of the original.There was no significant difference in cell survival rate,extracellular LDH and intracellular SOD activity in OMV4 group,and the highest activity of intracellular ACP,LDH and iNOS(P<0.05).The secretion of proinflammatory cytokines(TNF-?and IL-6)increased to 2-5 and 3-5 times,and the secretion of anti-inflammatory cytokines(IL-10 and TGF-?),was significantly higher than that of other groups.The cell survival rate,LDH and SOD activity in the OMV8 and OMV16 groups decreased significantly,and the secretion of proinflammatory cytokines(TNF-?and IL-6)increased to 9-15 and 10-17 times that of the original.(3)Escherichia coli-Bacillus subtilis shuttle plasmid YGJ32 containing fluorescent gene was used to transform Bacillus strain 168.The extracellular vesicles were isolated and purified from recombinant Bacillus subtilis.The recombinant Bacillus extracellular vesicles were observed under the fluorescence microscope,and the fluorescence was found.In summary,moderate BS-MOVs can enhance the immune function of macrophages by increasing the activity of macrophage immune related enzymes and regulating the secretions of cytokines. |
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