Preparation Of Time-Resolved Fluorescent Nanoparticles And Establishment And Application Of An Anaphylactic Reaction Evaluation Model

Objective:1.To produce a kind of fluorescent nanoparticles for time-resolved fluorescence immunoassay.2.To selecte a sensitive and stable model of mast cell degranulation in vitro by comparing the effects of different concentrations of Tween80 solution on the degranulation of RBL-2H3?P815 and Ku812 cells.And study the anaphylactic reactions of Yiqi Fumai Lyophilized Injection according to the selected cell model.Methods:1.Time-resolved fluorescent nanoparticles were prepared by emulsion polymerization and dispersion polymerization.In these reaction,the rare earth complex which was synthesized from rare earth element Europium and4,4,4-trifluoro-1-?2-naphthyl?-1,3-butanedione was used as luminescent substance and styrene was used as monomer.The particle size and dispersion of the nanoparticles were monitored by Ma Erwen particle size potential analyzer,the color and fluorescence intensity of the nanoparticles was respectively detected by UV lamp and Fluorescence spectrophotometer.2.Cultured RBL-2H3?P815 and Ku812 cells in vitro and determined the growth curve of three cells,stimulated cells which were in the logarithmic phase with different concentration Tween80 solution?0.04?0.2?1?5?10?20?40?80 mg/ml?,observed the staining effect of neutral red on three cells of different concentrations of Tween 80 solution by microscope,and calculated the percentage of degranulation.Meanwhile,detected the release of histamine by chemiluminescence assay,detected?-aminohexosaminidase by colorimetry and detected the release of trypsin by enzyme-linked immunosorbent assay?ELISA?.By comparing the difference of degranulation index among the three cell lines,a more suitable cell line was selected as the model to detect the degranulation of mast cells in vitro and investigate the anaphylactic reactions of Yiqi Fumai Lyophilized Injection.Furthermore,the IgE release of human mast cells stimulated by Tween 80 and Yiqi Fumai Lyophilized Injection respectively was also examined.Results:1.The rare earth complex which was synthesized from Eu³⁺and4,4,4-trifluoro-1-?2-naphthyl?-1,3-butanedione shows bright red color under ultraviolet light,which is the characteristic color of Eu³⁺.And the fluorescence spectrum of the rare earth complex showed the characteristic peak of Eu³⁺.The size of the nanoparticles prepared by emulsion polymerization is uniform,controllable and well dispersed?PDI<0.1?,but it is not colored under UV lamp.The nano-nanoparticles prepared by dispersion polymerization were red under UV lamp,the excitation spectrum showed the characteristic peak of Eu³⁺,but the dispersion was poor.After optimizing the reaction?adding appropriate amount of cross-linking agent during the reaction?,the particle size dispersion became better.2.In the three mast cell degranulation models,the percentage of degranulation,the release of trypsin,the release rate of histamine and?-aminohexanidase of three cells increased with the increase of concentration of Tween 80.In the same stimulation conditions,the degranulation effect of Ku812 cells was the most sensitive,but it was found that the stability and repeatability were poor.However,the sensitivity of RBL-2H3 cells was inferior to Ku812 cells,its stability and reproducibility were superior to Ku812 and P815 cells.While RBL-2H3 cells were used to investigate allergic and anaphylactic reactions of Yiqi Fumai Lyophilized Injection,finding it can causes cell degranulation.And the degree of degranulation was dependent on the concentration of Yiqi Fumai Lyophilized Injection.Making a comparison of the degree of degranulation stimulated by clinical equivalent concentrations of Yiqi Fumai Lyophilized Injection,Red ginseng,Radix Ophiopogonis and Schisandra chinensis,The degranulation indexes of Red ginseng,Radix Ophiopogonis and Schisandra chinensis were not higher than Yiqi Fumai Lyophilized Injection.But the degranulation degree of Radix Ophiopogonis was higher in three main components and was more close to the degree of degranulation of Yiqi Fumai Lyophilized Injection.In addition,the release of IgE by Tween 80 solution of 0.04-80 mg/ml or clinical equivalent concentrations of Yiqi Fumai Lyophilized Injection on Ku812 cells couldn't be detected.Conclusion:1.It has been proved that 4,4,4-trifluoro-1-?2-naphthyl?-1,3-butanedione as the ligand of rare earth element europium can effectively transfer energy to the central ion Eu³⁺and produce strong fluorescence.Emulsion polymerization is not suitable for the preparation of time-resolved fluorescent nanoparticles.While dispersion polymerization can make time-resolved fluorescent nanoparticles,finding cross-linking agent needs to be added in this reaction,and the amount of cross-linking agent has a great effect on the dispersion of the reaction system.The finding lays a foundation for further related research.2.The anaphylaxis induced by Tween 80 or Yiqi Fumai Lyophilized Injection may be anaphylactoid reaction.With the increase of Tween 80 concentration,three models were all degranulated significantly.Compared with Ku812 and P815 cells,RBL-2H3 cells were more suitable for degranulation detection model of mast cells in vitro.Yiqi Fumai Lyophilized Injection can cause anaphylactoid reaction,and Red ginseng,Radix Ophiopogonis,Schisandra chinensis as its main components,also can cause anaphylactoid reaction.And it was found that Radix Ophiopogonis is the main component which causes anaphylactoid reaction.That provides some theoretical guidance for the production and research department of Yiqi Fumai Lyophilized Injection.