| Propolis is the useful substance to maintain the health of the bee group.It can not only support the structure of the honeycomb,but also plays a decisive role in keeping internal environment.Antioxidant,anti-inflammatory,anti-bacterial,anti-tumor and other biological activities have been found in many modern researches about propolis.The component varies as the different origins,flavonoid contend has been widely used to assess the quality of propolis.Liposome is a spherical vesicle mainly composed of lipid bilayer and its structure is similar to the outer layer of cell.Therefore,the solubility of polar substances in the non-polar solvent can be greatly improved by being encapsulated in the liposome interior spherical structure or in the bilayer.Liposome can exert perfect slow release effect,reduce the toxicity of drugs and the targeting property can be also achieved after special modification.As a result,liposome can act as drug delivery carrier and potential adjuvant for vaccine with low irritation.The solubility of propolis flavonoids in aqueous solution can be largely enhanced,biological activities of propolis flavonoids and carrier effect of liposome can be synergistically utilized after propolis flavonoids being encapsulated in liposome.In this research,the microstructure of propolis flavonoids liposome,the sustained-release effect of propolis flavonoids liposome,the effects on spleen lymphocytes,peritoneal macrophages,dendritic cells and other immune cells were measured and evaluated to develop a modern veterinary drug product with a broad prospect.This research contains five parts as follows:Experiment 1 The preparation of propolis flavonoids liposome and its physical properties Propolis flavonoids liposome was prepared as the optimized condition.First of all,the particle size of Propolis flavonoids liposome was measured by particle size analyzer and the morphology of Propolis flavonoids liposome was observed by the transmission electron microscopy.Secondly,the encapsulation efficiencies of Propolis flavonoids liposome dissolved in PBS were determined at certain times lied on the amount of galangin and chrysin.Results showed that the particle size of Propolis flavonoids liposome was between 0.03?m to 1?m and the average value was about 100nm.Typical fingerprint-like structure could be seen under the electron microscope.Release profiles of propolis flavonoids liposome fitted first order linear relation and its half-life was about 4.Oh.Experiment 2 The effects of propolis flavonoids liposome on mouse spleen lymphocyte proliferation in vitro Five different concentrations of propolis flavonoids liposome,propolis flavonoids and blank liposome were added to mouse spleen lymphocytes in vitro individually,with PHA and with LPS,and the levels of mouse spleen lymphocyte proliferation were measured by MTT assay.Results showed that the proliferation of mouse spleen lymphocytes could be significantly actived by propolis flavonoids liposome and propolis flavonoids at concentrations of 3.75 ugˇmL-1 to 30 ugˇmL-1 and the effect of propolis flavonoids liposome was better than propolis flavonoids;The proliferation of mouse spleen lymphocytes could be significantly actived by propolis flavonoids liposome and propolis flavonoids with PHA at concentrations of 3.75 ugˇmL"1 to 30 ugˇmL-1,and the effect of propolis flavonoids liposome was better than propolis flavonoids at concentrations of 7.5 ugˇmL-1 to 30 ugˇmL-1;The proliferation of mouse spleen lymphocytes could be significantly actived by propolis flavonoids liposome and propolis flavonoids with LPS at concentrations of 3.75 ugˇmL-1 to 30 ugˇmL-1,and the effect of propolis flavonoids liposome was better than propolis flavonoids.Experiment 3 The effects of propolis flavonoids liposome on mouse myeloid dendritic cells Mouse bone marrow cells were collected and induced to DCs by rmG-CSFand rmIL-4.Seven days later,did experiments as follow:propolis flavonoids liposome were added to DCs for 48 hours,then DCs were co-culture with OVA and mouse spleen lymphocytes that immunized with OVA wrapped in FCA,evaluate antigen-present capability of DCs by MTT array;propolis flavonoids liposome were added to DCs for 48 hours,then DCs were co-culture with mouse spleen lymphocytes,measure the proliferation of mouse spleen lymphocytes by MTT array;propolis flavonoids liposome were added to DCs for 48 hours,examine positive expression rate of MHC-? CD80 and CD86 of DCs.Results showed that the antigen-present capability of DCs and mouse spleen lymphocytes proliferation could be enhanced by propolis flavonoids liposome,and the MHC-?.CD80 and CD86 expression could upregulated by propolis flavonoids liposome.Experiment 4 The effect of propolis flavonoids liposome on mouse peritoneal macrophages 6%starch broth was injected to mouse peritoneal and sacrificed to collect the peritoneal fluid two days later,then separated the peritoneal macrophage.Three different concentrations of propolis flavonoids liposome,propolis flavonoids and blank liposome were added to macrophages,the concentration of IFN-??IL-1??IL-6?IL-12 and TNF-a in the supernatant of macrophages were determined and phagocytic function of macrophages were assessed by Vybrant Phagocytosis Assay Kit after 48 hour's incubation.As the results showed,the secretion of IFN-??IL-laand IL-6 by macrophages were significantly improved by propolis flavonoids liposome,and the phagocytic function of macrophages were also enhanced.Experiment 5 The immune response of mouse immunized by OVA influented by propolis flavonoids liposome 120 5-mouse-old mouse were randomly divided to 5 groups and immunized by OVA subcutaneous,whose adjuvants were propolis flavonoids liposome,propolis flavonoids,blank liposome,FCAand NS respectively,and Immunized again two weeks later.At day 7,14,21,28,35 and 42 after first vaccination,blood were collected from ophthalmic vein,then four mouse were sacrificed to take spleens out under sterile environment and to prepare spleen lymphocytes,the concentrations of IgG?IL-4 and IFN-y were examined by ELISA kits and the proliferation of spleen lymphocytes respectively with PHA and LPS were measured by MTT array finally.As data illustrated,IgG?IL-2and IFN-? of immunized mouse the proliferation of mouse spleen lymphocytes were evidently upregulated at some times when propolis flavonoids liposome acted as adjuvant.To sum up,propolis flavonoids liposome was a potential adjuvant with broad prospects. |
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