| (Background and Objective] Chitin is homopolymer of beta-l,4-linked N-acetylglucosamine. Chitosan is the product of partial deacetylation of chitin They are the second most abundant polysaccharides in nature, comprising the horny substance in the exoskeletons of crabs, shrimp, and insects as well as fungi. Chitin and chitosan have been proved to be nontoxic, biodegradable, and biocompatible and are known to have various biological activities including antitumor activity, antimicrobial activity, antifunga) activity, immune-enhancing effects. However, their high viscosity and insolubility in neutral aqueous solutions restrict their uses in vivo Recent studies on chitosan have attracted 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. Oligochitosan has been observed to upregulate the production of interleukin-l(IL-l), tumor necrosis factor- a (TNF- a ), granulocyte macrophage colony stimylating factor(GMCSF), interleukin-6 (IL-6) by macrophage and induce immunologic adjuvant effects. Many aspects of oligochitosan's functions have been discovered by researchers, but the mechanism is still unclear.Our experiment observed the binding of fluorescence labeled oligochitosan with RAW264.7macrophage and explored the specific binding activity of oligochitosan with the component of macrophage. This work provides a foundation for further research on separation of combinding oligochitosan component of 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] l.Cell culture: RAW264.7, a murine macrophage-like cell line, were cultured in RPMI1640 medium with 10% heat-inactivated fetal bovine serum, 2mM L-glutamine, lOOU/ml penicillin and 100 u g/ml streptomycine at 37 掳C in a humidified atmosphere containing 5% CO2. 2.Label oligochitosan with 2-AMAC: 5 U 1 of an aqueous 20mM solution of oligochitosan was lyophilized in a microcentrifuge tube. A 10 1 sample of lOOmM solution of 2-aminoacridone(Molecular Probes) in glacial acetic acid /DMSO(3/17,v/v) and 10 l of 1M solution of NaCNBH3(Sigma) in tetrahydrofuran were added. The mixture was incubated at 45掳C for 5 hours then lyophilized, at last dissolved in 400 l DMSO/deionized water(l/l,v/v). Control assay, in which no oligochitosan was used, was carried out simultaneously. 3.Observe the binding of oligochitosan labeled with 2-AMAC with macrophages under confocal laser microscope and analysis the fluorescence intensity by flow cytometer using the Cell Quest software. [Result and Discussion] 1.Combination between 2-AMAC labeled oligochitosanand macrophage: 2-AMAC-oligochitosan first bound the cytomembrane of macrophage, and then diffused in the whole cytoplasm, at last entered the nucleolus and diffused in the whole cell. Fluorescence intensity increased with time. The internalization of oligochitosan in macrophage is a time-course process. 2.Initial experiments tested time, temperature, and concentration dependence of 2-AMAC-oligochitosan uptake in macrophages. Scatchard analysis of internalization of 2-aminoacridone-oligochitosan in macrophages indicated its internalization is mediated by a specific receptor on macrophage membrane with Ka of 2.1 10-5M. 3.Different oligochitosan fragments showed different effect on oligochitosan uptake, suggesting receptor recognition dependent on the structure of oligochitosan 4.Competition studies showed that mannose inhibited oligochitosan internalization, while LPS and P - glucan could not. 5.Inhibition of mannose-...
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