【作者】 冯洁；

【导师】 赵鲁杭；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 背景和目的 几丁质是由N-乙酰葡萄糖胺组成的多糖，它是世界上除纤维素之外含量最丰富的天然多聚物。壳聚糖是几丁质不完全脱乙酰的产物。几丁质和壳寡糖具有多种生物效应，包括抑瘤、抗菌和免疫增强等。但是它们的高粘度和在水溶液中的不溶性极大的限制了在体内的应用。于是低分子量、水溶性好、无毒且生物适应性好的壳寡糖逐渐得到了重视。 巨噬细胞(Macrophage)既是免疫细胞，又是辅佐免疫应答细胞，可通过吞噬、抗原呈递、分泌细胞因子、激活淋巴细胞和产生活性氧、NO等作用而行使抗感染、抗肿瘤、免疫应答和免疫调节作用，是机体免疫系统的重要组成部分。整体动物实验表明壳寡糖能激活巨噬细胞，增强巨噬细胞的杀伤活性，诱导如白介素-1(IL-1)、肿瘤坏死因子α(TNF-α)、粒单核集落刺激因子(GM-CSF)、白介素-6(IL-6)等细胞因子的分泌，增加T细胞和NK细胞的活性。但壳寡糖在巨噬细胞表面的受体及壳寡糖对巨噬细胞免疫功能的调节机理报道甚少。 本实验通过体外研究荧光标记壳寡糖与小鼠巨噬细胞的结合情况，揭示巨噬细胞壳寡糖结合组分的糖结合特异性，为进一步阐明壳寡糖对巨噬细胞的免疫调节机理和分离巨噬细胞壳寡糖结合组分提供一定的实验依据，为壳寡糖抗肿瘤药物和功能食品的开发提供理论依据。 材料和方法 1．RAW264.7巨噬细胞的培养：RAW264.7巨噬细胞于RPMI 1640培养液(加上10％灭活的小牛血清，2mmol／L L-谷氨酰胺，100U／ml青霉素和100U／ml链霉素)中培养。2．2-氨基吖啶酮标记壳寡糖：取5ul 20mmol／L壳寡糖于0.5ml Eppendorf管，离心冷冻干燥，加10ul 100mmol／L 2-氨基吖啶酮，10 ul 1.0mol／L硼氢氰化钠，混匀，离心。置于45℃水浴反应5小时，取出，离心冷冻干燥。3．在激光扫描共聚焦显微镜下观察2-氨基吖啶酮标记壳寡糖与巨噬细胞的结合，用流式细胞仪分析荧光强度。 结果和讨论 1．2-氨基吖啶酮标记的壳寡糖与巨噬细胞的结合情况：2-氨基吖啶浙江大学硕士学位论文酮标记的壳寡糖先与巨噬细胞膜有结合，然后分布于整个细胞质，最后进入细胞核，随时间的进展而呈现了一个内在化过程。2.2一氨基叮睫酮标记的壳寡糖与巨噬细胞的结合是一个依赖时间、温度和浓度的过程，Scatchard分析后发现它对巨噬细胞的作用是通过膜上特异性的受体(Kd为2.1 X 10一SM)进行的。3.不同的壳寡糖组成结构单位对巨噬细胞摄取壳寡糖产生不同的影响，说明壳寡糖的结构是受体识别的关键。4.竞争试验表明甘露糖能抑制壳寡糖的摄取，脂多糖、p-葡聚糖则不能，提示巨噬细胞壳寡糖结合组分可能为甘露糖受体。5.甘露糖一BSA、岩藻糖一BsA、N一乙酞葡萄糖胺一BsA都能抑制壳寡糖的摄取。结论1.壳寡糖可与巨噬细胞膜结合，并随时间的延长逐步进入巨噬细胞内。2.壳寡糖在巨噬细胞膜表面有特异性结合的受体(Kd为2.1 x 10一SM)，可能为一廿露糖受体。更多还原

【Abstract】 (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-更多还原