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Title: 不同基材與表面構型對肝細胞型態與活性之影響
The influence of various substrates and surface topographics on hepatocytic morphology and function
Authors: Ko-Liang Kuo
郭科良
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 初代肝細胞;半乳糖化基材;藥物篩選
Primary hepatocyte;galactosylated substratum;Drug-screening
Date: 2008
Issue Date: 2011-07-04
Abstract: 本論文的目的是欲建立一套有效性之體外(in vitro)藥物篩選系統來取代傳統的臨床動物藥檢模式。在此研究中,採用的方法是利用膠原蛋白(collagen)、果膠(pectin)等基質對具有微結構之PDMS進行基底塗層,另一方面則是藉由化學改質法將半乳糖嫁接至PDMS的表面上。之所以將PDMS表面半乳糖化的原因在於: 若初代肝細胞培養於表面半乳糖化之基材上,初代肝細胞會藉由asialoglycoprotein receptor (ASGPR) 接受器與半乳糖配體(ligand)產生專一性結合;經此模式與基材結合的初代肝細胞在型態上會呈現細胞球體(Spheroid)的構型,而在此構型下之初代肝細胞則能有效的提升與維持初代肝細胞之專一性功能與活性。 經由傅立葉轉換紅外線全反射光譜儀(FTIR-ATR) 與化學分析電子能譜儀(ESCA)等分析,可初步驗證PDMS確實經由化學修飾而產生表面半乳糖化。此外,我們利用初代肝細胞與人類肝癌細胞株HepG2來檢視特定基材對於細胞行為的影響。藉由F-actin與DAPI覆染的結果,可發現HepG2培養在不同基底塗層下,其細胞型態會隨著基質的不同而有所差異。在含有果膠(pectin)基底塗層與半乳糖化的PDMS上,細胞則是產生圓形或者是團聚的型態,而在含有膠原蛋白基底塗佈的PDMS中,則是發現細胞在型態上呈現了延展型(spreading)的變化。另一方面,藉由EROD assay我們發現在表面半乳糖化的PDMS上則可維持初代肝細胞CYP1A1/2之活性達一星期之久。
The purpose of this study is to develop an in vitro drug efficiency screening system for replacing the traditional animal model. In this study, the patterned-PDMS was coated with various substrates, including collagen and pectin. Besides surface coating, we also immobilized galactose on the PDMS surface since galactose was well known as ligand for ASGPR (asialoglycoprotein receptor) of primary hepatocyte. Through the specific ligand-receptor binding, primary hepatocytes could display the spheroid morphology which could maintain the liver cellular specific functions. From the results of Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) and electron spectroscopy for chemical analysis (ESCA) indicated that we had galactosylated of the surfaces of PDMS successfully. Furthermore, we utilized primary hepatocyte and HepG2 cells, a human hepatocellular liver carcinoma cell line, to examine the effects of specific substratum for the cellular behaviors. From the results of F-actin and DAPI staining, we found that HepG2 cells displayed circular morphology and cell-aggregates on pectin-coated and galactosylated PDMS, but expressed a spreading phenomenon on collagen-coated PDMS. Moreover, the result of EROD assay proved that galactosylated PDMS could maintain the CYP1A1/2 activity of primary hepatocyte more than one week.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M95360030
http://ntour.ntou.edu.tw/ir/handle/987654321/17730
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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