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研究生:蘇郁琇
研究生(外文):Yu-Hsiu Su
論文名稱:小鼠癌幹細胞研究
論文名稱(外文):A Study on Murine Cancer Stem Cells
指導教授:劉宏仁劉宏仁引用關係
指導教授(外文):Hung-Jen Liu
口試委員:楊明德廖采苓
口試委員(外文):Ming-Te YangTsai-Ling Liao
口試日期:2015-07-13
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:78
中文關鍵詞:癌幹細胞表面抗原瘤球癌幹細胞親和胜肽
外文關鍵詞:cancer stem celltumorosphereCSC surface markerCSC homing peptide
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自1994年證實人類急性骨髓性白血病(acute myelois leukemia, AML)之癌幹細胞後,乳癌、腦癌、肝癌、大腸癌、肺癌與胰臟癌,陸續被證實。2008年人類肺癌幹細胞的球狀培養方法出版後,人類乳癌、肝癌和大腸癌癌幹細胞的球狀培養方法相繼發表,目前已經是最為廣用的方法。Weiswald等呼籲以tumorosphere作為球狀幹細胞團的名稱。據此,我們開發了小鼠乳癌、大腸癌與肝癌之癌幹細胞的tumorosphere培養方法,後文中使用「瘤球」作為中文譯名。以cDNA microarray分析三組小鼠癌幹細胞/癌細胞之基因表現圖譜得知三種小鼠癌幹細胞相對癌細胞共同升調2倍以上的基因有166個;共同降調2倍以下的基因有28個。三種小鼠癌幹細胞除均表現已知CD44與E-Cadherin人類癌幹細胞表面標誌外,亦表現胚幹細胞表面標誌TRA-1-60、TRA-1-81、SSEA-1、SSEA-4與SSEA-5。利用以M13 phage display的方法,先去除和癌細胞結合的背景,篩選只會吸附到癌幹細胞的clones。經由DNA序列分析,目前已找到12組七胜肽。與TumorHoPe資料庫744種癌細胞親和胜肽序列比較,無明顯相似性,顯示我們所發現的12種七胜肽為全新的癌幹細胞親和性胜肽。

Since the first discovery of cancer stem cell (CSC) of AML (acute myeloid leu-kemia) in 1994,the identifications of CSCs were further demonstrated in breast can-cer,brain cancer,liver cancer,colon cancer,lung cancer and pancreatic cancer. Besides the self-renewal activity of normal stem cell, CSCs are resistant to chemotherapy, radi-otherapy, hypoxia and immune-surveillance. And CSCs are thought the major reason of cancer relapse.Simple suspension culture methods had been developed for human CSC of lung cancer, breast cancer, liver cancer and colon cancer. Tumorosphere was suggested to name the cell cluster in the suspension cultures. Based on these methods, suspension cultures for mouse breast, colon and liver CSCs were developed. The expression profiles of the three CSC/CC pairs were analyzed by cDNA microarray. There are 166 common up-regulated and 28 down-regulated genes among the three CSCs relative to CCs. All of the three murine CSCs express the known human CSC surface markers CD44 and E-Cadherin. The murine CSCs also express the embryonic stem cell (ESC) surface markers TRA-1-60,TRA-1-81,SSEA-1,SSEA-4,and SSEA-5. Hepta-peptides specific binding to mouse CSCs were screened by M13 phage display method. Phage library was pre-adsorbed with cancer cells to reduce the background of cancer cells. Twelve kinds of hepta-peptides were summarized from all of the clones sequenced. These peptide sequences are not similar in comparison to the 744 sequences in the TumorHoPe database. That is to say, our sequences are the novel CSC binding members.

中文摘要…………………………………………………………………………... i
Abstract………………………………………………..…………………………… ii
目次………………………………………………………………………………... iii
圖表目次………………………………………………………………………….. v
縮寫字對照表…………………………………………………………………….. vii
Chapter 1. 緒論………………………………………………………………….. 1
1.1.癌細胞與癌幹細胞………………………………………………………… 1
1.2.癌幹細胞瘤球模式…………………………………………………………… 2
1.3.腫瘤標靶胜肽………………………………………………………………… 3
1.4.圖表…………………………………………………………………………..... 5

Chapter 2. 癌幹細胞特有標誌…………………………………………………... 10
2.1.前言……………………………………………………………………………. 10
2.2.材料與方法……………………………………………………………………. 10
2.2.1.小鼠癌細胞培養方式………………..…………...…………………. 10
2.2.2.小鼠癌幹細胞表面抗原免疫螢光染色法…….……………………… 11
2.2.3. RNA萃取………………………………………………………...... 13
2.2.4. cDNA microarray晶片分析…….………………………………….. 13
2.2.5. RT-qPCR試驗…………………………………………………….... 14
2.3.結果與討論……………………………………………………………………. 14
2.3.1.小鼠癌細胞培養方式……………………..……………………………. 14
2.3.2.小鼠癌幹細胞表面抗原免疫螢光分析法……………………………… 15
2.3.3. cDNA microarray 晶片分析…..……………………………………… 15
2.3.4. RT-qPCR分析…………………………………………………………... 16
2.3.5.共同升調與降調基因與癌幹細胞的關係……………………………… 18
2.4.圖表……………………………………………………………………………. 19

Chapter 3. 篩選癌幹細胞特異性胜肽…………………………………………….. 36
3.1.前言……………………………………………………………………………. 36
3.2.材料與方法……………………………………………………………………. 36
3.2.1. M13 phage Ph.D.-7 library篩選EMT6 peptide library (第一階段)……. 37
3.2.2. EMT6 peptide篩選CT26、Hepa1-6、mESC特異性胜肽(第二階段)... 37
3.2.2.1. EMT6 peptide篩選CT26、Hepa1-6特異性胜肽................. 37
3.2.2.2. EMT6 peptide篩選mESC特異性胜肽……………………… 38
3.2.3.質體純化………………………………………………………………… 39
3.2.4.標靶癌幹細胞特異性胜肽螢光標定…………………………………… 39
3.3.結果與討論……………………………………………………………………. 40
3.3.1. EMT6 CSC homing peptide library……………………………………... 40
3.3.2.第二階段篩選…………………………………………………………… 40
3.3.2.1. CT26/EMT6 CSC homing peptide library…..…………………. 40
3.3.2.2. Hepa1-6/EMT6 CSC homing peptide library………………….. 40
3.3.2.3. mESC胚幹細胞篩選…………………………………………... 41
3.3.2.3.1. mESC/EMT6 CSC homing peptide library…….. 41
3.3.2.3.2. Free mESC/EMT6 CSC homing peptide library….. 41
3.3.3. CSC HP之分類…………...…………………………………………….. 41
3.3.4.胜肽合成螢光標定染色結果…………………………………………….. 42
3.4.圖表…………………………………………………………………………..... 44

Chapter 4. 展望…………………………………………………………..….….... 51
4.1.確定癌幹細胞表面共同特異性標誌………………………………………….. 51
4.2.癌幹細胞所篩選寡胜肽上的標的為何………………………………………. 51
4.2.1. Affinity chromatography………………………………………………... 51
4.2.2. Mass蛋白質身分鑑定………………………………………………..... 51
4.3.篩選出寡胜肽是否有特殊結構………………………………………………. 52
4.3.1. NMR…………………………………………………………………...... 52
4.3.2. X-ray diffraction………………………………………………………… 52
4.4.寡胜肽與藥物結合……………………………………………………………. 52
4.4.1. Antibody-drug conjugates (ADCs)…………………………………..….. 52

References………………………………………………………………………….. 55
Appendix I RNA QC圖譜 .…………………………………………………….….. 59
Appendix II .……………………………………………………………………...... 60
Appendix II-1癌幹細胞(EMT6 CSC)/癌細胞(EMT6 CC)表面抗原螢光分析圖 60
Appendix II-2癌幹細胞(CT26 CSC)/癌細胞(CT26 CC)表面抗原螢光分析圖… 64
Appendix II-3癌幹細胞(Hepa1-6 CSC)/癌細胞(Hepa1-6 CC)表面抗原螢光
分析圖…………………………………………………………....... 68
Appendix III……………………………………………………………………….. 72
Appendix III-1 EMT6(CC/CSC)特異性胜肽標靶癌幹細胞.................................. 72
Appendix III-2 CT26(CC/CSC)特異性胜肽標靶癌幹細胞……………………… 74
Appendix III-3 Hepa1-6 (CC/CSC)特異性胜肽標靶癌幹細胞…………………... 76
自我簡介…………………………………………………………………………... 78


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