Zhu Xin Hao, Gan Seng Keat, Wang Chi-Hwa, Tong Yen Wah
Department of Chemical and Biomolecular Engineering, National University of Singapore, 21 Lower Kent Ridge Road, Singapore 119077.
J Biomed Mater Res A. 2007 Dec 1;83(3):606-16. doi: 10.1002/jbm.a.31257.
The synergistic effects of extracellular matrix (ECM) protein combinations on Hep3B cell proliferation and functions are studied herein. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres were covalently conjugated with three types of proteins, collagen (type I), laminin, and fibronectin, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide cross linkers. Successful conjugations of protein molecules were verified by the presence of nitrogen peaks in X-ray photoelectron spectroscopy. The densities of grafted proteins were quantified using Micro-BCA kit. A human hepatoma cell line, Hep3B, was then cultured in vitro on the ECM proteins-modified microspheres for 2 weeks. Cell proliferation was estimated using MTT method, and two hepatic functions, albumin secretion and P-450 activity, were evaluated using ELISA and EROD assays, respectively. The results indicated that combination of the three ECM proteins on microsphere surfaces has a significant effect on the proliferation of Hep3B cells, thus better mimicking the in vivo environment for liver tissue engineering.
本文研究了细胞外基质（ECM）蛋白组合对Hep3B细胞增殖和功能的协同作用。使用1-乙基-3-（3-二甲基氨基丙基）碳二亚胺和N-羟基琥珀酰亚胺交联剂，将聚（3-羟基丁酸酯-co-3-羟基戊酸酯）（PHBV）微球与三种蛋白质，即I型胶原蛋白、层粘连蛋白和纤连蛋白进行共价偶联。通过X射线光电子能谱中氮峰的存在验证了蛋白质分子的成功偶联。使用微量BCA试剂盒对接枝蛋白的密度进行定量。然后将人肝癌细胞系Hep3B在体外培养于ECM蛋白修饰的微球上2周。使用MTT法评估细胞增殖，并分别使用ELISA和EROD测定法评估两种肝功能，即白蛋白分泌和P-450活性。结果表明，微球表面三种ECM蛋白的组合对Hep3B细胞的增殖有显著影响，从而更好地模拟了肝脏组织工程的体内环境。
