中国组织工程研究 ›› 2013, Vol. 17 ›› Issue (16): 2994-3001.doi: 10.3969/j.issn.2095-4344.2013.16.021

• 材料力学及表面改性 material mechanics and surface modification • 上一篇    下一篇

肝细胞特异性大孔微载体的制备与表征

郭伟,龚独辉,胡志伟,程远, 高毅,潘明新   

  1. 南方医科大学珠江医院肝胆二科,广东省广州市  510282
  • 收稿日期:2013-03-15 修回日期:2013-03-19 出版日期:2013-04-16 发布日期:2013-04-16
  • 通讯作者: 潘明新,博士,主任医师,教授,南方医科大学珠江医院肝胆二科,广东省广州市 510282 pmxwxy@sohu.com
  • 作者简介:郭伟★,男,1984年生,江西省吉安市人,汉族,南方医科大学在读硕士,主要从事非生物支架材料的研究。 921329794@qq.com
  • 基金资助:

    国家高新技术研究发展(863)计划项目(2006AA02A141),广东省科技计划项目(2007B010600009)。

Preparation and characterization of hepatocyte-specific macroporous microcarrier

Guo Wei, Gong Du-hui, Hu Zhi-wei, Cheng Yuan, Gao Yi, Pan Ming-xin   

  1. Second Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou  510282, Guangdong Province, China
  • Received:2013-03-15 Revised:2013-03-19 Online:2013-04-16 Published:2013-04-16
  • Contact: Pan Ming-xin, Doctor, Chief physician, Professor, Second Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong Province, China pmxwxy@sohu.com
  • About author:Guo Wei★, Studying for master’s degree, Physician, Second Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, Guangdong Province, China 921329794@qq.com
  • Supported by:

    he National High Technology Research and Development Program of China (863 Program), No. 2006AA02A141*; the Science and Technology Plan of Guangdong Province, No. 2007B010600009*

摘要:

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40-80 µm,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。

关键词: 生物材料, 材料力学及表面改性, 肝细胞特异性, 大孔微载体, 丝素蛋白, 细胞培养, 壳聚糖, 半乳糖基, 冷冻干燥法, 生物人工肝, 863项目

Abstract:

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 well-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 cell biocompatibility test were used to characterize its morphology, physical and chemical properties.
RESULTS AND CONCLUSION: The microcarrier was successfully 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 successfully compounded into the scaffold material which was confirmed by the proton nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. Cell biocompatibility test showed a hepatocyte-specific affinity to the silk fibroin/galactosylated chitosan macroporous microcarrier, and the cells 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.

Key words: biomaterials, material mechanics and surface modification, hepatocyte-specific characteristics, macroporous microcarrier, silk fibroin, cell culture, chitosan, galactosyl, freeze-drying, bioartificial liver, National “863&rdquo, Project of China

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