肝细胞特异性大孔微载体的制备与表征**★

摘要

BACKGROUND:Hepatocyte culture is the key to the hepatocyte transplantation and bioartificial liver study. It has turned into the first problem how to get adequate number of better activity and wel-functioning hepatocytes. OBJECTIVE:To prepare a high-performance silk fibroin/galactosylated chitosan macroporous microcarrier with hepatocyte-specific characteristics, for hepatocyte adhesion culture in the three-dimensional environment in vitro. METHODS:Silk fibroin, chitosan, and lactose acid served as raw materials to prepare a hepatocyte-specific silk fibroin/galactosylated chitosan macroporous microcarrier using comprehensive application of emulsified-chemical crosslinking, polarity solution and freeze-drying. Optical microscope, scanning electron microscope, proton nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, and cel biocompatibility test were used to characterize its morphology, physical and chemical properties. RESULTS AND CONCLUSION:The microcarrier was successful y prepared in the project. Furthermore, the pores were occupied inside and outside of the microcarrier and interconnected, which presented hepatocyte-specific characteristics. Under the scanning electron microscope, the macroporous microcarrier exhibited a trumpet-shaped porous structure with the pore size of 40-80 µm and even pore distribution, providing an appropriate environment for high-density culture of hepatocytes. The galactosyl was successful y compounded into the scaffold material which was confirmed by the proton nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. Cel biocompatibility test showed a hepatocyte-specific affinity to the silk fibroin/galactosylated chitosan macroporous microcarrier, and the cel s grew and aggregated around the microcarrier exhibiting a good activity. These findings indicate that combination of emulsified-chemical crosslinking, polarity solution and freeze-drying can prepare a silk fibroin/galactosylated chitosan macroporous microcarrier with hepatocyte-specific characteristics.%　　背景：在肝细胞移植及生物人工肝的研究中，肝细胞培养是关键环节，肝细胞表面存在着去唾液酸糖蛋白受体和半乳糖基之间的分子识别功能，如何方便地获取足够数量、活性较好、功能良好的肝细胞已经成为其首要问题。　　目的：制备一种新的体外三维环境下具有肝细胞特异性的高性能的大孔微载体。　　方法：以丝素蛋白、壳聚糖及乳糖酸为基本材料，通过乳化-化学交联、冷冻干燥、极性溶液处理制备一种新型丝素蛋白/半乳糖基化壳聚糖大孔微载体。采用光学显微镜、扫描电子显微镜和通过超导核磁共振波谱、傅里叶变换红外光谱及细胞相容性等对其形态结构及理化性质进行表征。　　结果与结论：实验所制备的丝素蛋白/半乳糖基化壳聚糖大孔微载体，内外多孔且相互连通，具有肝细胞特异性。扫描电子显微镜下观察显示，该大孔微载体表面呈多孔结构，孔径为40-80µm，开口向外，呈喇叭状，孔的分布均匀，内部亦为多孔结构，适合于肝细胞高密度培养。超导核磁共振波谱、傅里叶变换红外光谱检测结果显示，半乳糖基已经成功复合到支架材料之中。细胞相容性实验结果显示，肝细胞能与材料特异性黏附、细胞围绕微载体聚集生长，并表现出很好的活力。结果证实，应用乳化-化学交联、极性溶液处理、冷冻干燥等方法可制备出具有肝细胞特异性亲和力的丝素蛋白/半乳糖基化壳聚糖大孔微载体。