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研究生:郭科良
研究生(外文):Ko-Liang Kuo
論文名稱:不同基材與表面構型對肝細胞型態與活性之影響
論文名稱(外文):The influence of various substrates and surface topographics on hepatocytic morphology and function
指導教授:許富銀
指導教授(外文):Fu-Yin Hsu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:91
中文關鍵詞:初代肝細胞半乳糖化基材藥物篩選
外文關鍵詞:Primary hepatocytegalactosylated substratumDrug-screening
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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.
目錄

誌謝 ……………………………………………………………….………III
中文摘要 IV
英文摘要 VI
目錄 ………………………………………………………………………VII
圖目錄 X
表目錄 XII
第一章: 前言 1
1.1 藥物開發的簡介 1
1.2 研究動機 3
第二章: 文獻回顧 5
2.1 肝臟簡介 5
2.2 藥物代謝 (Drug metabolism) 8
2.3 藥理試驗簡介 11
2.4 體外(in vitro)肝細胞藥物篩選模式 12
2.5 臨床藥檢模式動物的選擇 18
2.6 肝細胞體外(in vitro)培養系統 18
2.7 膠原蛋白(collagen)與果膠(Pectin)之簡介 22
2.8 PDMS (polydimethylsiloxane) 23
第三章: 實驗材料 25
3.1 實驗用藥品 25
3.2 實驗用耗材與儀器 28
3.3 實驗動物與細胞株 29
第四章: 實驗方法 30
4.1 Patterned PDMS 之設計 32
4.2製備non-patterened與patterened PDMS 33
4.2.1 Non-patterened PDMS 33
4.2.2 Patterened PDMS 33
4.2.3製備Galactosylated PDMS 33
4.3 Chemical modified PDMS 鑑定方法 35
4.3.1 Amino-grafted PDMS 鑑定: TNBS assay 35
4.3.2 Galactosylated PDMS鑑定: 35
4.3.3 FTIR-ATR與ESCA分析半乳糖化PDMS 36
4.4 Patterned & Non-patterned PDMS表面基質塗層 36
4.5 WST-1 assay 37
4.6 大鼠初代肝細胞分離 38
4.7 F-actin/DAPI counterstain 40
4.8 CYP 1A1/2活性測定 (EROD assay) 41
第五章: 結果與討論: 42
5.1 矽晶圓母模與 Patterned-PDMS尺寸大小之鑑定 42
5.2 PDMS之化學改質與鑑定: 44
5.2.1 PDMS表面胺基化反應 44
5.2.2 PDMS表面半乳糖化 45
5.2.3 PDMS表面半乳糖化之定量分析: 51
5.3 肝細胞分離 55
5.4 HepG2細胞型態的改變: 57
5.5 WST-1 assay 60
5.6 EROD assay : CYP1A1/2表現情形 62
5.7 F-actin/DAPI stain 68
第六章:結論 75
第七章 參考文獻(Reference) 78

圖目錄
圖一、肝臟內部基本結構 5
圖二、肝細胞於不同配體(ligand)間型態上的差異 20
圖三、實驗基本流程 31
圖四、光學輪廓儀之檢測 42
圖五、掃描式電子顯微鏡(SEM)之檢測 43
圖六、PDMS經胺基化反應前後的外觀 44
圖七、以TNBSA assay檢測胺基化反應之結果 45
圖八、全反射式傅立葉轉換紅外線光譜……………………………….48
圖九、Lactobionic acid之標準曲線圖 51
圖十、ESCA: 化學分析電子光譜 53
圖十一、 初代肝細胞分離之結果圖 56
圖十二、 不同基質與表面微結構對於Hep G2細胞型態之影響 59
圖十三、Hep G2於不同基材之WST-1細胞活性分析 61
圖十四、初代肝細胞在第二天於不同基質上EROD的表現情形 64
圖十五、初代肝細胞在第四天於不同基質上EROD的表現情形 65
圖十六、初代肝細胞在第八天於不同基質上EROD的表現情形 66
圖十七、初代肝細胞在第四天於Patterned-PDMS之EROD assay 67
圖十八、Hep G2培養八小時後進行F-actin/DAPI染色之結果 73

表目錄
表一、人體CYP450種類與性質 11
表二、 Pristine PDMS 之 FTIR-ATR 50
表三、ESCA圖譜分析各材料之元素比例 54
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