BP016W-新型食道鱗狀上皮細胞癌候選藥物｜國立中央大學博碩士論文系統

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研究生：	賴家祥 Chia-Hsiang Lai
論文名稱：	BP016W-新型食道鱗狀上皮細胞癌候選藥物 BP016W- as a Novel Candidate Drug for Esophageal squamous Cell Carcinoma Therapy
指導教授：	蘇立仁
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 系統生物與生物資訊研究所 Graduate Institute of Systems Biology and Bioinformatics
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	102
中文關鍵詞：	食道鱗狀上皮細胞癌、天然藥物、轉錄子定序、蛋白質質譜、生物資訊、降血脂藥物、細胞程序性凋
外文關鍵詞：	Esophageal squamous cell carcinoma, Traditional Chinese Medicine, Transcriptome sequencing, Protein mass, Bioinformatics, Hypolipidemic drug, Apoptosis
相關次數：	點閱：16 下載：0
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食道鱗狀上皮細胞癌是一個致命的癌症，它有高死亡率及嚴重的化療副作用。因此，我們決定找出新的食道癌治療藥物。我們首先分析食道鱗狀上皮細胞癌患者之轉錄子定序及基因微陣列晶片資料，並從中篩選了727個轉錄子(363上調轉錄子及364下調轉錄子)。接著，使用基因分析工具及藥物預測工具發現到它們的結果與降血脂藥物相關。近年來許多文獻以及臨床研究顯示天然藥物可以用於治療癌症，它有著低毒性並且可以抗發炎及抑制癌症細胞的生長。於是，我們搜尋有關降血脂的天然藥物並且從實驗中看到BP016W可以抑制癌細胞的生長。我們利用流式細胞儀看到BP016W抑制癌細胞的方式是促使癌細胞走向程序性凋亡路徑，接著我們使用西方墨點法證明細胞程序性凋亡蛋白被啟動。為了探討更全面的基因路徑表現以及蛋白質體學代謝表現，我們使用轉錄子次世代定序與蛋白質質譜儀來觀察癌細胞治療前後基因及蛋白質表現量差異。從高通量數據我們發現BP016W治療癌細胞可以抑制脂肪代謝及細胞生長路徑，這樣的結果表示我們之前的預測方法是正確的。從過去的文獻指出抑制脂肪代謝可以促使細胞走向程序性凋亡。所以，我們認為BP016W可以當作新的食道癌治療後選藥物。

Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal tumors in the world. It has low cure rate and has serious side effects of chemotherapy. We analyzed 15 patients’ transcriptome sequencing and microarray data selecting 727 transcripts (363 up- and 364 down-regulated) significant to the ESCC, finally. Then we put these signatures into LINCScloud database and the results indicated that hypolipidemic related drugs might be a novel approach for ESCC therapy. Traditional Chinese medicines (TCMs) originated in ancient China and has evolved over thousands of years. Many previous studies have indicated these bioactive natural products can inhibit the interaction, promotion, and progression of carcinogenesis in human cancer cells, and have low toxicity. So, the old book about the TCM was found that there were 4 herbs which related to our prediction. In the study, we screened 4 TCMs and found that BP016W, one of the families of Nelumbo, shows strong growth inhibition toward the ESCC cell line CE81T, thus was chosen for further study. Our western blotting and flow cytometry data both confirmed the term of apoptosis. The protein mass and transcriptome sequencing data also showed that the cholesterol was inhibited and could affect the mitochondria to cause the cell to be apoptosis. Eventually, we think that BP016W is a novel candidate for ESCC therapy.

Table of contents

中文摘要……………………………………………………………………………………......i
Abstract……………………………………………………………………………………...…ii
誌謝………………………………………………………………………………………...…iii
Table of Contents…………………………………………………………………………...…iv
List of Figures…………………………………………………………………………………vi
List of Tables…………………………………………………………………………………viii
List of Supplements Figures………………………………………………………………...…ix
List of Supplements Tables……………………………..…………………………………........x
1. Introduction………………………………………………………………………………….1
2. Materials and Method……………………………………………………………………….3
2-1. Cell culture………………………………………………………………………………...3
2-2. Preparation of Folium Nelumbinis……………………………………………………...…3
2-3. Preparation of Lovastatin………………………………………………………………….3
2-4. Cell viability assay………………………………………………………………………...4
2-5. Apoptosis and cell cycle analysis…………………………………………………………4
2-6. Preparation of protein extract……………………………………………………………..5
2-7. Western blotting…………………………………………………………………………...5
2-8. Preparation of RNA extract……………………………………………………………….6
2-9. Data analysis………………………………………………………………………………6
2-10. HPLC analysis of different fractions…………………………………………………….6
3. Result………………………………………………………………………………………..8
3-1. Bioinformatic analysis…………………………………………………………………….8
3-1-1. Transcriptome sequencing & functional analysis………………………………………8
3-1-2. Microarray analysis of Taiwanese ESCC patients…………………………………….13
3-2. Lovastatin caused ESCC’s cell line to be apoptosis……………………………………..15
3-2-1. In vitro cytotoxicity study by Alamar blue assay……………………………………...15
3-2-2. Analysis of apoptosis by cell cytometry……………………………………………….17
3-2-3. Expression of apoptotic pathway proteins by western blot……………………………18
3-2-4. Transcriptome sequencing analysis of CE81T treated with lovastatin………………..19
3-3. Hypolipidemic drug in Traditional Chinese medicine…………………………………...21
3-4. Folium Nelumbinis (BP016W) caused ESCC’s cell line to be apoptosis………………..23
3-4-1. BP016W suppressed ESCC’s cell line growth...............................................................23
3-4-2. Western blot verified pathway of apoptosis…………………………………………...25
3-4-3. Transcriptome sequencing analysis of CE81T treated with BP016W………………...27
3-4-4. Protein mass analysis of CE81T treated with BP016W……………………………….29
3-5. Active compound of fraction from BP016W…………………………………………….32
3-5-1. The isolated method of BP016W……………………………………………………...32
3-5-2. HPLC analysis…………………………………………………………………………35
4. Discussion………………………………………………………………………………….37
5. Reference…………………………………………………………………………………..42
6. Supplements………………………………………………………………………………..44
 

                                

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