1.The Preparation Of Chitosan Oligomer And Binding Of Oligochitosan In Macrophages 2.Separation And Purification Of Receptor On Macrophage Membrane That Binds The Oligochitosan

[Background and Objective] Chitin is one of the most abundant biological natrue resources, comprising the horny substance in the exoskeletons of crabs, shrimp , and insects as well as fungi . Chitin as well as its partially deacetylated product-chitosan , have many kinds of physiological functions .including antitumor activity,antimicrobial activity, antifungal activity, immuno-enhangcing effects. However, their high insolubility and high viscosity in neutral aqueous solutions restrict their application. Recent studies on chitosan have attract interest for oligochitosan which is not only water-soluble, non-toxic, biocompatible, but also possess versatile functional properties .Macrophage is a kind of immune cell as well as accessory cell. It can function as antiinfection, anticancer, immune response and immunoregulation by phagocytosis, antigen presentation, cytokines secretion, lymphocyte activation , reactive oxygen and NO production. It' s an important part of body immune system.Our experiment observed the binding of fluorescence labeled oligochitosan in RAW264. 7 macrophage . This work provides a foundation for further research on separation of combinding oligochitosan componentof macrophage and elucidation on the immunoregulation mechanism of oligochitosan with macrophage. From this work we endeavor to find the theoretical ground for development of antitumor drugs and functional food from oligochitosan.[Methods] 1. Chitosan was hydrolyzed with enzyme, then the chitosan oligomer was extrated with organic solvent, following by purifying the chitohexose by chromatograph method and detecting the chitosan oligomer component by HPLC. 2. Cell culture: RAW264. 7 , amurine macrophage-like cell line , were cultured in RPMI 1640 medium with 10 % heat-inactivated fetal bovine serum , 2 mM L-glutamine , 100 U / ml penicillin and 100μg / ml streptomycine at 37 °C in a humidified atmospere containing 5% CO₂. 3. Label mannan with TRITC: 5 mg of TRITC were dissolved in 0.25 ml N, N dimethy-L formamide, then ethyleneglycol was added to the solution. 20 mg of mannan were dissolved in the mixture of 1ml ethyleneglycol and 3ml sodium carbonate buffer(PH 9.3), and the solution was mixed with the TRITC solution. The final solution was keeped from light and dialyzed with the solution of PBS (0.01M, PH 7. 4 ) entill there was no fluorescence found in the outside buffer, then the TRITC-mannan solution were lyophilized. 4. Observe the bingding of TRITC labled mannan with macrophages under confocal laser microscope and analysis the fluorescence intensity by flow cytometer using the Cell Quest software.[Result] 1. The chitosan oligomer in which the main component was 6⁷ oligomer could be produced by preparation technology. 2. Combination between TRITC labled mannan and macrophage: TRITC-mannan first bound the cytomembrane of macrophage, and then diffused in the whole cytoplasm, at last diffused in the whole cell, and the bingding process was a concentration-time dependent one. 3. Cometition studies: Just like other inhibitions of the mannose receptor, oligochitosan could inhibitthe mannan internalization, and this inhibitory effect was as high as 44% .[Conclusions] Oligochitosan could bind to macrophage, and the binding process is seemed to carried out through the mannose receptor on the membrane of macrophage.