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研究生:林美瑤
研究生(外文):Mei-Yao Lin
論文名稱:建立篩檢藥物回復人類子宮頸癌細胞接觸抑制的細胞分析法
論文名稱(外文):Development of a cell-based assay for screening chemicals restoring HeLa cell contact inhibition
指導教授:蔡有光
指導教授(外文):Yeou-Guang Tsay
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:58
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現代抗癌藥物的研發,於早期開發階段特別著重在分子標靶的研究,例如癌蛋白質。一般認為,這些癌蛋白質的結構和功能,應該是篩選小分子化合物或生物分子的基本資訊。然而,幾十年下來,癌蛋白質數目龐大,分子標靶藥物卻寥寥可數,如此的數量懸殊,顯然證明這類藥物開發手段非常沒有效率。基於這些理由,我們決定嘗試以表現型做為藥物篩選的基本原則。接觸抑制是當細胞周圍均與其他細胞接觸後,細胞的移動性和有絲分裂的速率會大幅度地降低,是一種使細胞的生長維持在單層細胞厚度的機制。由於培養的癌細胞,仍保有癌組織逃避接觸抑制的特性,我們發展出一個篩檢藥物平台,以回復癌細胞接觸抑制作為分析目標。透過優化平台實驗條件,我們選擇合適的Alexa螢光染劑和培養環境,讓高培養密度的HeLa細胞,確實有較高含量的新合成DNA。同時結合Hoechst染色,我們從一個化合藥物庫中,篩選出19個有可能回復HeLa細胞接觸抑制特性的化合物。此外,我們透過19個化合物歸類出的藥理學特性,也找出調節細胞生長的新機制。
Modern discovery of anti-cancer therapeutics emphasizes the significance of molecular targets, i.e. oncoproteins, in the early process, since these targets should be used as the structural and functional basis for screening of chemicals or biologics. However, such an approach has proven very ineffective considering the glaring discrepancy between the numbers of oncoproteins and target therapeutics. Because of these problems, we proposed that phenotype-based assays are better systems in drug screening than those target-based ones. Contact inhibition is a growth mechanism, which functions to keep cells growing into a layer one cell thick, causing a dramatic decrease of cell mobility and mitotic rate in confluent cells. Since bypass of contact inhibition is a biological property seen both in cancer tissue and in culture cells, we propose to establish assays to test whether any compounds might have the activities to restore contact inhibition property in cancer cells. Following some optimization, we selected adequate Alexa Fluor dyes and incubation vessels, which helps show that HeLa cells in dense culture indeed have high amount of nascent DNA synthesis. In combination with Hoechst staining, we found that a group of nineteen compounds from a chemical library exhibits the potential to restore contact inhibition. Furthermore, the pharmacological properties of these classes of compounds may shed light on new mechanisms for cell growth regulation.
TABLE OF CONTENTS
致謝……………………………………….………...………………………………….I
中文摘要……………………………………….………...…………………………..III
ABSTRACT……........................................................……………………………….IV
TABLE OF CONTENTS……………………………………….………...……...……V
LIST OF TABLES……………………………………….………...……………...…VII
LIST OF FIGURES……………………………………….………...……………...VIII
LIST OF ABBREVIATIONS…………………………………………………….......IX
CHAPTER I LITERATURE REVIEW…………………………..……………………1
Introduction of anti-cancer therapeutics discovery……………………………….2
Contact inhibition……………………………………….………...……………...8
CHAPTER II Development of a cell-based assay for screening chemicals restoring HeLa cell contact inhibition…...……………………………………………………...14
SUMMARY…………………………………………………………………......15
INTRODUCTION………………………………………………………....……16
MATERIALS AND METHODS………………………………………………..18
RESULTS……………………………………………………………………….20
DISCUSSION…………………………………………………………………..35
REFERENCES…………………………………………………………………….....39
SUPPLEMENT…………………………………………………………….………...44

LIST OF TABLES
CHAPTER II
Table 1. The structures of nineteen candidates each of which can restore HeLa cell contact inhibition…………………………..............................................................31
Table 2. A list of nineteen candidates each of which can restore HeLa cell contact inhibition……..…………………………................................................................33
SUPPLEMENT
Table 1. A list of fluorescent signals of Hoechst 33342 and Alexa Flour 555 upon incubation of one of the 786 drug compounds in confluent HeLa cells………………44

LIST OF FIGURES
CHAPTER II
Figure 1. The assay to screen compounds that can restore HeLa cell contact inhibition…………………………………………………………......................21
Figure 2. Fluorescent staining of confluent HeLa cells stained by Alexa Fluor® 647 dye or Alexa Fluor® 555 dye……………..…………………………...........23
Figure 3. Fluorescence microscopy of confluent HeLa cells stained by Alexa Fluor® 647 dye or Alexa Fluor® 555 dye……………………………………….24
Figure 4. Fluorescence microscopy of different culture densities in HeLa cells……………………………………………...................................................26
Figure 5. Fluorescence microscopy of different culture densities in HeLa cells………………………………………...........................................................27
Figure 6. Screening of compounds that can restore HeLa cell contact inhibition with our cell-based assay.…………………………………..................................28
Figure 7. Comparison with the efficacy of click chemistry with or without trypsinization in 30% confluent HeLa cells………………………......................36
Figure 8. Comparison with the efficacy of click chemistry with or without trypsinization in 100% confluent HeLa cells……………………........................37

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