博碩士論文 972211007 詳細資訊




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姓名 楊庭(Ting Yang)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 大腸癌細胞株之 EGFR—K-ras 訊號路徑的基因微陣列實驗 與化學基因體學分析
(Analysis of EGFR—K-ras pathways through microarrayconjunction with chemical genomics in colon cancer cell lines )
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摘要(中) 了解和分析複雜的基因調控-表達-回饋的過程,必頇以系統生物學觀念,藉由運用高通量檢
測及資訊分析等方法來完成。微陣列實驗及其高通量檢測技術興起,為系統生物學奠定了
一個重要的里程碑。
本研究利用外顯子微陣列晶片(Exon array)高通量基因分析實驗,篩選 Sw480、Caco2、Ht29
細胞株經由 EGF 刺激和 Cetuximab 阻斷後,對於 EGFR 下游外顯子基因的啟動或其它路徑
的變化基因。利用 SAM 的生物晶片顯著分析方法,篩選出 3052 個有顯著意義變化的基因,
並做基因功能分類以及經 KegArray 軟體處理對應到 KEGG 路徑的列表,找出各細胞株之
間相對的特異性。再利用 R 套件畫出熱相圖並用歐氏距離和 Pearson 相關係數計算出每株
細胞控制組和實驗組之相似性。
經由 KegArray 聯結 KEGG 生物訊息路徑資料初步比對後,發現 Sw480、 Ht29 、Caco2
細胞株經 Cetuximab 阻斷 EGFR 之後於 EGFR-Ras 路徑的差異性以及不同下游子生物路徑
的改變情形。
未來在我們研究當中,希望可以針對大腸直腸癌細胞經 Cetuximab 阻斷 EGFR-Ras 路徑後
之整體訊息路徑變化以及相關關鍵基因進行分析,並觀看其各子路徑中的基因變化。希望
可以藉由系統生物學的概念整合出關鍵基因在不同的生物路徑中複雜的關係與交互作用,
並探討基因分子訊息路徑與上下游基因之調控機制。
摘要(英) In order to understand the complexity of regulation, expression and feedback mechanism of
genes, the methodology of system biology through the high-throughput analysis and information
analysis must be utilized to elucidate the gene regulation network. The advance in microarray
experiment and its high throughput analysis has set up an important milestone in the system
biology.
In this study, we used the exon array and high throughput data analysis software developed
by Partek Corporation to observe the change of EGFR downstream targets after EGF and
Cetuximab blockade in Sw480, Caco2 and Ht29 cells. By using SAM biochip significance
analysis method, 3052 significantly different genes were selected. These genes were grouped by
different functions and relativity between different cell lines by correlating these genes with
KEGG mapping .We analyze the heat map of each cell line before and after stimulation by using
R package. We also compare the difference of control and experiment group and similarity
between other two groups by using Euclidean distance and Pearson correlation.
Using KegArray in conjunction with KEGG biological information pathway data,we found
the different downstream pathway transformation.of the Sw480、 Ht29 、Caco2 cell line after
treatment with Cetuximab to block EGFR and EGFR-Ras pathway.
In the furture, we hope to detect different gene mutation after treated with Cetuximab using
colon cancer cell lines and observe gene alteration in various EGFR-Ras pathway. We want to
integrate complex gene interaction by using system biology. It is valuable to study and
investigate the regulation mechanism of upstream and downstream interaction in a pathway.
關鍵字(中) ★ 大腸直腸癌細胞
★ 生物路徑
關鍵字(英) ★ EGFR
★ KEGG
★ K-ras
論文目次 v
目 錄
中文摘要 ..................................................................................................................... i
Abstract ...................................................................................................................... ii
致 謝 ............................................................................................................... iii
中英專有名詞對照表 ............................................................................................... iv
圖目錄 ....................................................................................................................... vi
第一章 緒 論 ......................................................................................................... 1
1-1 前言 ........................................................................................................... 1
1-2 生物路徑重要性與意義 ........................................................................... 1
1-3 目前常用觀察生物路徑之工具................................................................... 2
1-3-1 生物資料庫 ....................................................................................... 2
1-3-2 生物資料庫應用之軟體 .................................................................... 3
1-3-3 KEGG 和 KegArray ........................................................................... 3
1-4 本實驗所採用之細胞訊號傳遞模式 ....................................................... 4
1-4-1 大腸直腸癌與 EGFR、K-ras 基因突變 ........................................... 4
第二章 實驗材料與方法 .......................................................................................... 7
2-1 藥品與材料 .................................................................................................. 7
2-1-1 儀器.................................................................................................... 7
2-2 大腸直腸癌細胞株 SW480、 CACO2 、HT29 之培養與處理 ............. 8
2-3 K-RAS 突變檢測 .......................................................................................... 9
2-3-1 DNA 萃取 ........................................................................................... 9
2-3-2 聚合酶連鎖反應 ................................................................................ 9
2-3-3 K-ras 突變基因檢測 .......................................................................... 9
2-4 外顯子晶片實驗設計與流程 ..................................................................... 10
2-4-1 RNA 樣品準備 ................................................................................. 10
2-4-2 Ribosome RNA 移除 ........................................................................ 11
2-4-3 cDNA 反轉錄 ................................................................................... 11
2-4-4 cDNA 片段化 ................................................................................... 11
2-4-5 單股 cDNA 標識(lable) ................................................................... 12
2-5 資料分析 ................................................................................................. 12
2-5-1 晶片輸出資料之初階分析 ............................................................. 12
2-5-2 生物晶片顯著性分析(Significance analysis of microarray(SAM))
.................................................................................................................... 13
2-5-3 叢集(clustering) ................................................................................ 14
2-5-4 基因表現於 KEGG 資料庫網路分析 ............................................. 14
vi
第三章 結 果 ...................................................................................................... 15
3-1 細胞生長情形與刺激後結果.................................................................... 15
3-2 EGFR 染色體基因於各細胞株的表現 ..................................................... 15
3-3 生物晶片分析結果 ..................................................................................... 15
3-3-1 晶片結果的初階評估 ...................................................................... 15
3-3-2 晶片資料叢集分析結果 ................................................................. 15
3-3-3 各細胞株特異性的叢集與參與重要路徑 ..................................... 16
第 四 章 討 論 .................................................................................................... 17
4-1 三株細胞對 Cetuximab 阻斷後型態變化 ................................................ 17
4-2 EGFR 染色體基因定量 ............................................................................. 17
4-3 生物晶片分析結果及生物意義................................................................. 18
第五章 結 論 .......................................................................................................... 20
Reference .................................................................................................................. 21
參考文獻 21
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指導教授 凌慶東(Qing-Dong Ling) 審核日期 2010-7-15
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