臺灣博碩士論文加值系統

前言及目的：近來研究顯示第二型環氧化酶(英文全名, COX-2)在人類細胞的癌化過程中扮演相當重要的角色，COX-2與癌症之細胞生長及腫瘤形成有關。此外，我們了解到複方療法的評估在藥物學上是一個相當重要的問題，特別應用在癌症的化學治療上。大部份的癌症化學治療，會運用一或兩種以上的化學治療劑，目的在減少抗藥性及副作用並且提高療效。本研究的目的，在探討利用其第二型環氧化酶選擇性抑制劑Etodolac來抑制肝癌細胞株Hep G2及HA22T，而且可配合抗癌化學藥物5-FU的使用，以提高療效，推論此作用可能是經由加強誘導細胞凋亡所達成。我們考慮藥物的交互作用，以合併給藥及接續給藥的不同方式，來探討Etodolac合併5-FU，對肝癌細胞株Hep G2及HA22T的細胞生長抑制情形。
材料與方法：本實驗使用肝癌細胞株HepG2及HA22T及正常細胞株KEL FIB來進行Etodolac, 5-FU及Etodolac合併5-FU的複方療法實驗，將藥物分別加入細胞株中作用48小時，分別以MTT (3-(4,5– dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)細胞毒性試驗法及流式細胞分析儀(flow cytometry)來分析細胞生長的抑制情形及細胞凋亡的現象。
實驗結果：Etodolac或5-FU，對肝癌細胞株HepG2及HA22T及正常細胞株KEL FIB有細胞生長抑制作用，而且呈劑量及時間依存性關係。另外在流式細胞分析儀的分析中，發現Etodolac及5-FU可引發肝癌細胞株HepG2及HA22T之細胞凋亡，也發現Etodolac在與5-FU的合併給藥中會誘導細胞凋亡的增加，而且在兩藥合併给藥的細胞凋亡分析中，同時給藥法會優於接續給藥的方法。
結論：第二型環氧化酶選擇性抑制劑Etodolac在5-FU毒殺肝癌細胞的化學治療上扮演輔助的功效，在這些研究中，Etodolac合併5-FU的細胞凋亡現象以合併給藥的方法效果最為顯著，這些發現提供一個臨床前治療的試驗參考，可提供在5-FU對於肝癌化學治療上扮演複方療法的選擇。

Introduction and purpose : Recent studies have demonstrated that cyclooxygenase-2 (COX-2) plays a crucial role in tumorigenesis, COX-2 is usually cited to be involved in tumorigenesis of cancer. As well known to all, the evaluation of synergistic effects is one of the most issue in pharmacological studies. Especially, in cancer therapy, most chemotherapeutic agents are administered with a single or two other drugs with the aim to reduce the drug resistance, side effects and to increase the tumor suppresive rate. The aim of this study was to investigate whether the selective COX-2 inhibitors (e.x., etodolac) exert anti-proliferative effect in hepatocellular cancer cell lines Hep G2 and HA22T and may be used in combination with the conventional chemotherapeutic drugs 5-FU for the treatment of hepatocellular cancer cell lines, the latter effect is possibly mediated via enhancement of apoptosis. Thus we investigated the interactive effects of a selective COX-2 inhibitor, etodolac, in combination with 5-FU in hepatocellular cancer cell lines, in simultaneous or sequential administration schedules.
Materials and Methods : Hepatocellular cancer cell lines of Hep G2, HA22T and KEL FIB cell lines were cultured. The selective COX-2 inhibitor etodolac, 5-FU, or etodolac combined with 5-FU were added to the cultures and co-cultured for 48 hours respectively. Cell proliferation and apoptosis rate were observed with MTT assay and flow cytometry.
Results : Both etodolac and 5-FU inhibited growth of hepatocellular cancer cell lines and KEL FIB cell line in a dose-dependent and time-dependent manners. The apoptotic activity induced by etodolac in combination with 5-FU was more effective than etodolac or 5-FU alone as evidenced by the cell death of hepatocellular cancer cell lines. Furthermore, the apoptotic rate induced by etodolac in combination with 5-FU was significantly increased compared to that by sequential exposure to etodolac then to 5-FU or 5-FU then followed by etodolac.
Conclusion : Etodolac, a selective COX-2 inhibitor can be used as a subsidiary drug in 5-FU chemotherapy for treating hepatocellular cancer cell lines. In our study, we have demonstrated that the cytotoxic effect of etodolac, based on the induction of apoptosis and inhibition of proliferation and differention, in combination with 5-FU was schedule-dependent, which is actually favoring a simultaneous administration. These findings provide a preclinic experiment basis for a combinatorial therapy using etodolac and 5-FU in treatment of human hepatocellular carcinoma.

Contents
Table of Abbreviations.....................................1
KeyWords...................................................2
Chapter 1、Abstract (摘要).................................3
1.1、Chinese Abstract (中文摘要)...........................3
1.2、English Abstract (英文摘要)…..............................................................4
Chapter 2、Introduction (前言)...............................................................................6
2.1、Hepatocellular Carcinoma (HCC)………………………...................6
2.2、Chemotherapy Preventation of Hepatocellular Carcinoma..............6
2.3、5-FU (附圖一) and 5’DFUR/Capecitabine………………..…………...…..7
2.4、Cyclooxygenases-2 (COX-2).......................................................7
2.5、Preferential COX-2 Selective Inhibitor, Etodolac..........................8
2.6、Cell Signals and Apoptosis....................................................................10
Chapter 3、Background of The Study (研究背景)..................................................16
Chapter 4、Materials and Methods (材料與方法)..................................................18
4.1、Cell Lines and Culture Methods............................................................18
4.2、Drugs...................................................................................................18
4.3、MTT Assay...........................................................................................18
4.4、DNA Fragmentation and Trypan Blue Exclusion Assay for Cell Death ..19
4.5、Annexin V assay for Relocated Phosphatidylserine...............................20
Chapter 5、Results (結果).....................................................................................22
5.1、Dose and Time-dependent Inhibitory Effects on Growth Following
Treatment with 5-FU or Etodolac…………………………………............22
5.2、Dose Responsive Interaction between Etodolac and 5-FU.......................22
5.3、Dose-dependent Induction of DNA Fragmentation by TUNNEL Assay....22
5.4、Induction of Apoptosis by Etodolac in Combination with 5-FU
Administered Simultaneously or Sequentially by Annexin-V Assay.........23
Chapter 6、Discussion (討論)..............................................................................26
Chapter7、Conclusion (結論)…………..………………………………………..……26
Chapter 8、Reference (文獻)................................................................................27
Figures.................................................................................................................36
附件一…………………………………………………………………………..…….…48
附件二…………………………………………………………………………...………49

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