【作者】 刘利；

【导师】 万涛；

【作者基本信息】 武汉理工大学 ， 生物医学工程， 2007， 硕士

【摘要】 肝脏是人体的重要器官，是人体内物质代谢的中心，它具有代谢、排毒、循环调节等功能。然而，肝癌、急性肝衰竭（脂肪肝、肝硬化、肝炎）等疾病导致了大量的死亡，严重威胁人类的健康，所以研制理想的肝组织工程支架材料是医学和生物材料科学领域中的重要课题。壳聚糖是甲壳素的脱乙酰化产物，是生物相容性和生物可降解性好，安全无毒的天然聚合物，它是一种独特的碱性多糖，在结构中壳聚糖分子链吡喃环上的活性基团-氨基和羟基，可接枝不同的化学基团进行改性，制备不同的衍生物。肝细胞是一种依赖贴壁性细胞，其表面存在着脱唾液酸糖蛋白受体，而半乳糖则是肝细胞表面脱唾液酸糖蛋白受体的特异性配体，是肝细胞识别的相应位点并能产生特异性相互作用。通过引入含有半乳糖的残基，壳聚糖可以增加肝细胞的附着点。肝细胞在体内是生长在一个由各种细胞外基质构成的三维交联网络中，在体外这种帖壁依赖的细胞则需要附着在材料上，才能生长和代谢，因此我们从改善材料的化学结构和几何形态角度考虑，模拟体内的三维环境，制备出半乳糖化的多孔壳聚糖用于肝细胞的培养，以提高细胞的培养密度和代谢活性。本研究论文以天然聚合物壳聚糖，乳糖酸为原料，通过EDC（1-乙基-3-（3-甲氨丙基）碳亚胺盐）和NHS（N—羟基琥珀酰亚胺）交联剂活化，使壳聚糖分子中的NH₂与乳糖酸的羧基反应，冷冻干燥，制备出多孔的半乳糖化壳聚糖，利用红外光谱、核磁共振、XRD、DSC，接触角对合成的物质进行定性，定量以及理化性质的测定，结果确证壳聚糖的2-NH₂上引入了半乳糖基，且取代度为11.62％，XRD，DSC对其物理性质进行了分析，数据显示壳聚糖经改性后，由于氢键能力的降低，导致结晶度和热稳定性的降低，亲水性变好。采用冷冻干燥技术制备了三维多孔半乳糖化壳聚糖支架，对制备的三维多孔支架的理化性能和细胞相容性进行了检测，并将肝细胞在三维多孔半乳糖基化壳聚糖支架和薄膜上进行了共培养。结果表明：由冻干法制备的三维多孔支架材料在不同冷冻温度下形成的孔径大小不一，在-20℃和-70℃下的冷冻温度下所制的支架孔径主要分布在30μm-200μm，而在-196℃的冷冻温度下所制的支架孔径主要分布在10μm-50μm；不同温度制备的支架材料的孔隙率、吸水率、保水率、压缩强度等都有所不同；半乳糖化壳聚糖的细胞增殖能力高于壳聚糖支架，没有溶血现象。肝细胞与其支架共培养显示半乳糖的引进使细胞培养密度，和代谢活性得到了提高。更多还原

【Abstract】 Liver is one of the most important and complex organs, serving several indispensable functions such as metabolism, elimination of toxicant, regulation of circulation in the body. Currently, many patients with liver failure are suffering from loss of liver functions. So the research of optimal liver scaffold became the urgent task of medicine and biological material fields. Chitosan as a binary heteropolysaccharide containing linked 2-acetamide-2-β-D-glucopyranose and 2-amino-2-deoxy-β-D-glucopyranose residues has characteristic similar to glycosaminoglycans and is biodegradable and non-toxic. The chitosan molecule has amino and hydroxyl groups which can be modified chemically. Hepatocytes are anchorage-dependent cells on which surfaces asialoglycoprotein receptors exist, however galactose is known as a specific adhesive ligand to asialoglycoprotein receptor (ASGR) of hepatocyte. The introduce of the galactose residues could strengthen the anchorage site within the scaffold and promote the specific cell interactions and various cell fuctions including migration, proliferation, and specific gene expression. Hepatocyte in vivo grows in the three dimensions and cross-link network composed of various ECMs and in vitro this anchorage-dependent cells must adhere to materials to grow and metabolism.So from the point of view of improving chemical structure of materials and geometry size,we mimic the vivo three-dimension environment and prepare the porous galactosylated chitosan scaffold to culture hepatocyte for promoting the culture density and the metabolic activation.In this paper, the porous galactosylated chitosan was synthesized by the reaction the amino group of chitosan and of carboxyl group of lactobionic acid under activiation of EDC and NHS; freeze-drying technique. The structure and physical behaviors were characterized by FT-IR,~1HNMR DSC, XRD, water contact angle and the morphology and properities were analysed. The results confirm the introduction of galatose in the position of 2-NH2 of chitosan and that after the modification of chitosan,because of the decline of hydrogen bond capacity, their degree of crystallinty and thermal stability reduce. Three-dimensional galactosylated chitosan scaffolds for tissue engineering were fabricated by freezing and lyophilizing methods.The physicochemical properties and the biocompatibility of mimic three-dimensional scaffolds were studied and the hepatocytes were cultured in galactosylated chitosan scaffolds. The results shows that the pore diameters under the cool temperature of-20℃and-70℃ranges from 30um to 200um however the pore diameters under the cool temperature of-197℃ranges from 10um-30um,the porous scaffolds which prepared under different temprature have different porosity, water absorption rate, retention rate, compressive strength. The cell proliferation capcity of galactosylated chitosan scaffolds was stronger than chitosan scaffolds and the galactosylated chitosan scaffold don’t exist the hemolysis phenomenon.The co-culture of hepatocyte and galactosylated chitosan scaffolds showed that because of the introduce of galactose residue, the hepatocyte density and metabolic activity were greatly improved.更多还原