臺灣博碩士論文加值系統

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

Robbins and Cotran Pathologic Basis of Disease, 7th ed, p. 878
Strain AJ, Neuberger JM. “A bioartificial liver - State of the art”. Science. 2002;Vol. 295:1005-1009
Okazaki I, Ninomiya Y, Kyuichi T , and Friedman SL. “Extracellular Matrix and the Liver: Approach to Gene Therapy”. Academic Press. 2003；Ch. 1: 15-17
Stevens A, Lowe, J. S. “Human histology”. Elsevier/Mosby. 2005；3rd ed Ch. 12:229-238
Handschin C, Meyer UA. “Induction of drug metabolism: The role of nuclear receptors”. Pharmacological Reviews. 2003;Vol. 55:649-673.
Kwon, Younggil. “Handbook of Essential Pharmacokinetics, Pharmacodynamics and Drug Metabolism for Industrial Scientists” Springer US. 2002;Ch. 8:121-168
FJ Gonzalez and YH Lee. “Constitutive expression of hepatic cytochrome P450 genes”. FASEB Journal. 1996;Vol.10:1112-1117
Paivi Taavitsainen. “Cytochrome P450 isoform-specific in vitro methods to predict drug metabolism and interactions”. Oulu University Press
Leucuta, Sorin E.; Vlase, Laurian. “Pharmcokinetics and metabolic drug interactions”. Current clinical pharmacology. 2006;Vol. 1: 5-20
Hans Gerhard Vogel. “Drug Discovery and Evaluation Safety and Pharmacokinetic Assays”. Chapter II. J. Drug-Drug Interaction Enzyme-Induction
Albert P Li.“Preclinical in vitro screening assays for drug-like properties”. Drug Discovery Today Technologies. 2005;Vol. 2:179-185
Rane A, Wilkinson GR, Shand DG. “Prediction of Hepatic Extraction Ratio from Invitro Measurement of Intrinsic Clearance”. Journal of Pharmacology and Experimental Therapeutics. 1977;Vol. 200:420-424.
Maurel P. “The use of adult human hepatocytes in primary culture and other in vitro systems to investigate drug metabolism in man”. Advanced Drug Delivery Reviews. 1996;Vol. 22 :105-132.
Park, Kevin; Williams, Dominic P; Naisbitt, Dean J; Kitteringham, Neil R; Pirmohamed, Munir. “Investigation of toxic metabolites during drug development”. Toxicology and Applied Pharmacology. 2005;Vol. 207:S425 – 434
Baranczewski, Pawe?; Sta?czak, Andrzej; Kautiainen, Antti; Sandin, Per; Edlund, Per-Olof. “Introduction to early in vitro identification of metabolites of new chemical entities in drug discovery and development”. Pharmacological Reports. 2006;Vol. 58:341-352
M.D. Tingle, and N.A. Helsby l. “Can in vitro drug metabolism studies with human tissue replace in vivo animal studies?” Environmental Toxicology and Pharmacology. 2006;Vol. 21:184-190
Ekins, S, Ring, BJ, Grace, J, McRobie-Belle, DJ, Wrighton, SA. “Present and future in vitro approaches for drug metabolism”. Journal of Pharmacological and Toxicological Methods. 2000;Vol. 44:313-24.
Marc Vermeir , Pieter Annaert , Rao Nvs Mamidi , Dirk Roymans , Willem Meuldermans , Geert Mannens. “Cell-based models to study hepatic drug metabolism and enzyme induction in human”. Expert Opinion on Drug Metabolism and Toxicology. 2005;Vol. 1:75-90
Phillips, Ian R. and Shephard, Elizabeth A. “Ctytochrome P450 Protocols”. Publisher: Humana Press. 2nd chapter 26
Alpini G, Phillips JO, Vroman B, Larusso NF. “Recent advances in the isolation of liver cells”. Hepatology. 1994;Vol. 20:494-514
Funatsu, K; Ijima, H; Nakazawa, K; Yamashita, Y; Shimada, M; Sugimachi, K. “Hybrid Artificial Liver Using Hepatocyte Organoid Culture”. Artificial Organs. 2001;Vol. 25:194–200
Pelkonen O, Kaltiala EH, Larmi TKI and Karki NT. “Cytochrome P-450-linked monooxygenase system and drug-induced spectral interactions in human liver microsomes”. Chem-Biol Interact. 1974;Vol. 9: 205-216
De Kanter, R; Monshouwer, M; Meijer, DKF; Groothuis, GMM. “Precision-Cut Organ Slices as a Tool to Study Toxicity and Metabolism of Xenobiotics with Special Reference to Non-Hepatic Tissues”. Current Drug Metabolism. 2002;Vol. 3:39-59
Inoue, Y; Miyazaki, M; Tsuji, T; Sakaguchi, M; Fukaya, K; Huh, N H; Namba, M. “Reactivation of liver-specific gene expression in an immortalized human hepatocyte cell line by introduction of the human HNF4alpha2 gene”. International journal of molecular medicine. 2001;Vol. 8:481-487
Werner A, Duvar S, Muthing J, Buntemeyer H, Lunsdorf H, Strauss M, Lehmann J “Cultivation of immortalized human hepatocytes HepZ on macroporous cultispher G microcarrier”. Biotechnology and Bioengineering. 2000;Vol. 68:59-70
Andrews J, Abd-Ellah MF, Randolph NL, Kenworthy KE, Carlile DJ, Friedberg T. “Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial, yeast and human lymphoblastoid cells”. Xenobiotica. 2002;Vol. 32:937-947.
Barham HM, Lennard MS, Tucker GT. “An Evaluation of Cytochrome-P450 Isoform Activities in the Female Dark Agouti (Da) Rat - Relevance to Its Use as a Model of the Cyp2d6 Poor Metabolizer Phenotype”. Biochemical Pharmacology. 1994;Vol. 47:1295-1307.
Guillouzo A. “Liver cell models in in vitro toxicology”. Environmental Health Perspectives. 1998;Vol. 106:511-532.
Lewis DFV, Ioannides C, Parke DV. “Cytochromes P450 and species differences in xenobiotic metabolism and activation of carcinogen”. Environmental Health Perspectives. 1998;Vol. 106:633-641.
Zuber R, Anzenbacherova E, Anzenbacher P. “Cytochromes P450 and experimental models of drug metabolism”. Journal of Cellular and Molecular Medicine. 2002;Vol. 6:189-198.
Curtis ASG, Forrester JV, McInnes C, Lawrie F. “Adhesion of Cells to Polystyrene Surfaces”. Journal of Cell Biology. 1983;Vol. 97:1500-1506.
LeCluyse EL, Bullock PL, Parkinson A. “Strategies for restoration and maintenance of normal hepatic structure and function in long-term cultures of rat hepatocytes”. Advanced Drug Delivery Reviews. 1996 ;Vol. 22:133-186.
Du YN, Chia SM, Han RB, Chang S, Tang HH, Yu H. “3D hepatocyte monolayer on hybrid RGD/galactose substratum”. Biomaterials. 2006 ;Vol. 27:5669-5680.
MN Berry,Anthony M. Edwards .” The hepatocyte review”. Chapter 8
Gutsche, Annie Tang; Lo, Hungnan; Zurlo, Joanne; Yager, James; Leong, Kam W. “Engineering of a sugar-derivatized porous network for hepatocyte culture”. Biomaterials. 1996;Vol. 17:387-393
J.P. Chen, S.C. Yu, B.R.S. Hsu, S.H. Fu and H.S. Liu. “Loofa sponge as a sacffod for the culture of human hepatocyte cell line”. Biotechnology Progress. 2003;Vol. 19:522-527
Cho, CS; Seo, SJ; Park, IK; Kim, SH; Kim, TH; Hoshiba, T; Harada, I; Akaike, T. “Galactose-carrying polymers as extracellular matrices for liver tissue engineering”. Biomaterials. 2006;Vol. 27: 576-585
Ying, L., Yin, C., Zhuo, R. X., Leong, K. W., Mao, H. Q., Kang, E. T., and Neoh, K. G. “Immobilization of galactose ligands on acrylic acid grafted-copolymerized poly(ethylene terephthalate) film and its application to hepatocyte culture”. Biomacromolecules. 2003;Vol. 4:157-165
Kim, Sang-Heon; Kim, Jong-Hun; Akaike, Toshihiro. “Regulation of cell adhesion signaling by synthetic glycopolymer matrix in primary cultured hepatocyte”. FEBS letters. 2003;Vol. 553:433-439
Landry J, Bernier D, Ouellet C, Goyette R, Marceau N. “Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities”. Journal of Cell Biology. 1985;Vol. 101: 914-923
Abu-Absi SF, Friend JR, Hansen LK, Hu W-S. “Structural polarity and functional bile canaliculi in rat hepatocyte spheroid”. Experimental cell research. 2002;Vol. 274:56-67
Hay, E.D., ed. (1991) Cell Biology of Extracellular Matrix. New York: Plenum Press. 2nd .
Thakur, B.R.; Singh, R.K.; Handa, A.K.”Chemistry and uses of pectin--a review.” Critical reviews in food science and nutrition. 1997;Vol. 37: 47-73..
Folch, A; Toner, M, “Microengineering of cellular interactions”, Annual review of biomedical engineering. 2000;Vol. 2:227–256
G.M. Whitesides, E. Ostuni, S. Takayama, X.Y. Jiang and D.E. Ingber, “Soft lithography in biology and biochemistry”. Annual review of biomedical engineering. 2001; Vol. 3:335–373
Lemmo, AV; Rose, DJ; Tisone, TC, “Inkjet dispensing technology: applications in drug discovery”. Current Opinion in Biotechnology. 1998 ;Vol. 9:615–617
Kidambi, S; Udpa, N; Schroeder, SA; Findlan, R; Lee, I; Chan, C , Christina Chan “ Cell Adhesion on Polyelectrolyte Multilayer Coated Polydimethylsiloxane Surfaces with Varying Topographies.”. Tissue engineering. 2007;Vol. 13:2105-2117
Williams, RL; Wilson, DJ; Rhodes, NP “Stability of plasma-treated silicone rubber and its influence on the interfacial aspects of blood compatibility”.Biomaterials. 2004;Vol. 25: 4659-4673
Fukuda, Junji; Khademhosseini, Ali; Yeo, Yoon; Yang, Xiaoyu; Yeh, Judy; Eng, George; Blumling, James; Wang, Chi-Fong; Kohane, Daniel S; Langer, Robert. “ Micromolding of photocrosslinkable chitosan hydrogel for spheroid microarray and co-cultures”. Biomaterials. 2006;Vol. 27:5259-5267
Goda, Tatsuro; Konno, Tomohiro; Takai, Madoka; Moro, Toru; Ishihara, Kazuhiko. “ Biomimetic phosphorylcholine polymer grafting from polydimethylsiloxane surface using photo-induced polymerization”. Biomaterial. 2006;Vol. 27:5151–5160
Alpini G, Phillips JO, Vroman B, Larusso NF. “ Recent Advances in the Isolation of Liver-Cells”. Hepatology. 1994;Vol. 20:494-514.
Guodong Sui, Jinyi Wang, Chung-Cheng Lee, Weixing Lu, Stephanie P. Lee, Jeffrey V. Leyton, Anna M. Wu, and Hsian-Rong Tseng. “Solution-Phase Surface Modification in Intact Poly(dimethylsiloxane) Microfluidic Channels”. Analytical Chemistry. 2006;Vol. 78:5543-5551
Kuijpers, A.J., Engbers, G.H.M., Krijgsveld, J., Zaat, S.A.J., Dankert, J., Feijen, J.. “Cross-linking and characterisation of gelatin matrices for biomedical applications”. Journal of Biomaterials Science, Polymer Edition. 2000;Vol. 11: 225–243
Yin, C; Liao, K; Mao, H-Q; Leong, KW; Zhuo, R-X; Chan, V.”Adhesion contact dynamics of HepG2 cells on galactose-immobilized substrates”. Biomaterials. 2003;Vol. 24:837–850
Maxime A G, Antoine Q H C L, Borders Jr. “Stability of water-soluble carbodiimides”. Alalytical Biochemistry. 1990;Vol. 184;244-248
Brown HH. “A study of 2,4,6-Trinitrobenzenesulfonic acid for automated amino acid chromatograph”. Clinic Chemistry. 1968; Vol. 14:967–978
Seglen, P.O. “Preparation of rat liver cells Ⅱ. Effect of ions and chelators on tissue dispersion.” Experimental Cell Research, 1973; Vol. 76:25-30
Mary C. Phelan. “Technique for mammalian cell tissue culture”. Current Protocols in Neuroscience. 2007;Vol. 1:3B.1-A.3B.18
Evangelista MB, Hsiong SX, Fernandes R, Sampaio P, Kong HJ, Barrias CC. “Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix”. Biomaterials. 2007 ;Vol. 28:3644-3655.
Yin C, Chia SM, Quek CH, Yu HR, Zhuo RX, Leong KW. “Microcapsules with improved mechanical stability for hepatocyte culture”. Biomaterials. 2003;Vol. 24:1771-1780.
Kobayashi T, Saitoh H, Fujii N. “Porous Polydimethylsiloxane Membranes Treated with Aminopropyltrimethoxysilane. Journal of Applied Polymer Science. 1994;Vol. 51:483-489.
Bodas D, Khan-Malek C. “Formation of more stable hydrophilic surfaces of PDMS by plasma and chemical treatments”. Microelectronic Engineering. 2006;Vol. 83:1277-1279.