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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59633完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳惠文(Huei-Wen Chen) | |
| dc.contributor.author | Hsueh-Hua Chen | en |
| dc.contributor.author | 陳雪華 | zh_TW |
| dc.date.accessioned | 2021-06-16T09:30:50Z | - |
| dc.date.available | 2022-02-24 | |
| dc.date.copyright | 2017-02-24 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-02-16 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59633 | - |
| dc.description.abstract | 腫瘤幹細胞(Cancer stem cells, CSCs)是腫瘤細胞中，特別具有自我更新、腫瘤細胞分化、腫瘤起始能力以及抗藥性的亞群。因此，開發出針對腫瘤幹細胞的標靶藥物被認為是治療癌症新策略的關鍵。腫瘤幹細胞的維持主要是藉由在腫瘤微環境中腫瘤相關纖維母細胞作為餵養細胞，餵養細胞能壯大腫瘤幹細胞群體並促進腫瘤幹細胞相關基因的表現。腫瘤幹細胞與腫瘤相關纖維母細胞共培養系統可有助於腫瘤幹細胞標靶藥物的篩選。然而，須由病人檢體培養的腫瘤相關纖維母細胞，其來源受個體差異而影響腫瘤幹細胞及藥物篩選的效力與再現性。本研究中，目的是要建立一個無餵養細胞的腫瘤幹細胞培養系統。基於轉錄體學與細胞激素的蛋白質維陣列分析，我們從腫瘤幹細胞與腫瘤相關纖維母細胞共培養後收集到的細胞共培養液所含的細胞激素分析中，找出了幾個細胞激素，這些細胞激素存在於腫瘤微環境中，能有助於促進與維持腫瘤幹細胞的幹性基因。隨後，我們試圖從這些細胞激素中，以單獨及多個組合的方式，找到能維持腫瘤幹細胞特性的細胞激素組合。我們發現IGF-1結合IL-6可以顯著增加腫瘤球數和促進Nanog基因表達，這種組合也可以改善腫瘤幹細胞的透明質酸立體細胞培養盤。我們的結果說明通過使用IGF-1和IL-6細胞激素組合來維持腫瘤幹細胞特性的無餵養細胞培養系統是可行的，而此系統有助於建立腫瘤幹細胞的標靶藥物篩選系統。 | zh_TW |
| dc.description.abstract | Cancer stem cells (CSCs) are sub-populations of tumor cells with the properties of self-renewal, clonal tumor initiation ability, drug-resistance, and for tumor initiation. Hence, targeting CSCs has been suggested as the key for development of new treatment strategy to cure cancer. CSCs reside in the microenvironment, which are majorly supported via the cancer-associated fibroblasts (CAF), as the feeder cells. The feeder cells could enrich the CSCs population and promote the stemness-associated gene expression in the CSC populations. The co-culture system is helpful to establish a model for CSC-targeting drug screening. However, the sources of CAFs and the heterogeneity of such stromal cells might affect the CSC and the drug-screening efficacy and reproducibility. Here, we wanted to setup a feeder-free culture system for CSC. Base on the CSCs/CAFs co-culture system, we have identified the CSCs-dependent micro-environmental factors, which may enrich and maintain their “stemness” through identifying the cytokine profiling in the co-culture condition medium. Then, we tried to find the suitable combination of CSCs-dependent cytokines for establishing a feeder free culture system of lung CSCs. Base on the transcriptomic analysis and protein cytokine array, several candidate cytokines were performed to examine the individual and combinatory effects on maintaining the CSCs characteristics. ry to find the suitable combination of different cytokines for establishing a feeder free culture system of lung CSCs. Here, we found IGF-1 combined with IL-6 could significantly increase the tumorous sphere numbers and promote Nanog gene expression. Such combination could also improve the CSCs 3-D culture in the HA-chitosan Plates. Above all, our results show the promising to maintain CSCs in the feeder-free culture system by using IGF-1 and IL-6 combination. This model could be helpful for establish the drug screening system for targeting on CSCs. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T09:30:50Z (GMT). No. of bitstreams: 1 ntu-106-R03447010-1.pdf: 7922682 bytes, checksum: 2648b4bf85423178b75f81e0b69a1dcf (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書 .......................................................................................................................... i 致謝 .................................................................................................................. ii 中文摘要 ........................................................................................................... iii ABSTRACT ....................................................................................................... v 目錄 .................................................................................................................. vii 圖索引 ............................................................................................................... ix 表索引 ............................................................................................................... x 第一章 緒論……................................................................................................... 1 1.1 居高不下的肺癌死亡率…………….................................................................... 1 1.2 腫瘤幹細胞(Cancer stem cell)的發現…………………………..………………………….... 1 1.3 腫瘤微環境(Tumor microenvironment) ....................................................... 3 1.4 抗癌藥物篩選………………………………………...................................................... 4 1.5 幾丁質(Chitosan)、透明質酸(Hyaluronic acid, HA)與CD44…........................ 6 1.6 實驗目的……….............................................................................................. 8 第二章 材料與方法………...................................................................................... 10 2.1 實驗材料...................................................................................................... 10 2.1.1 肺癌細胞株................................................................................................ 10 2.1.2 藥品與試劑……………................................................................................... 11 2.1.3 儀器與設備............................................................................................... 13 2.2 實驗方法………………………………………............................................................. 13 2.2.1 分離共培養的腫瘤幹細胞與腫瘤相關纖維母細胞…………………………................ 13 2.2.2 收集Co-culture medium/Condition medium……………………………………..….. 14 2.2.3 細胞毒性試驗……………………………………………………………………………………...… 14 2.2.4 測試的細胞激素組別與濃度與給予細胞激素後的基因表現實驗流程................15 2.2.5 Sphere forming assay………………………………………………………………………..… 15 2.2.6 統計方法…………………………………………………………………………………………….... 16 第三章 結果………………………............................................................................... 16 3.1. 初步挑選可能影響腫瘤細胞幹性的細胞激素(cytokine) ……………………….......... 16 3.2. 給予挑選出來的細胞激素(Cytokine)是否能誘發腫瘤的幹細胞特性(Stemness) ………………………………………………………………………………………………………................. 17 3.3. 挑選出的細胞激素對腫瘤幹細胞生長特性(Tumorigenicity)的影響………........... 17 3.4. 蛋白質表現結果…………………………………………………………………………………….…...20 3.5. 細胞外基質的影響………………………………………………………………………………..…… 20 3.6. 3D culture………………………………………………..……………………………….……………. 22 第四章 結論……………………………………………………………………………………………….……. 23 第五章 結論與未來展望………………………………………………………………………………...…. 26 第六章 參考文獻……………………………………………………………………………………………..…29 圖附錄 圖1.1 螢光顯微鏡下的UGM細胞株…………………………………………………………............. 37 圖2.1 分離共培養的腫瘤幹細胞與腫瘤相關纖維母細胞…………………………................ 38 圖3-1. 從腫瘤相關纖維母細胞與腫瘤幹細胞共培養前後的基因微陣列(Affymatrix Gene Microarray)數據中挑選出的十六個基因，在兩種細胞共培養前後的變化…………….... 39 圖3-2. 以即時定量聚合酶連鎖反應分析給予細胞激素刺激後，幹細胞基因Nanog表現 ....……………………………………………………………………………………….……………............…. 41 圖3-3. 以sphere formation測試細胞激素對幹細胞特性的影響………………….......….. 43 圖3-4. 不同的肺癌細胞株A549、H1975及 CLS1的sphere formation實驗來測試細胞激素組合………………………………………………………………………………………………............…. 44 圖3-5. 調整IGF-1、IGF-2與IL-6細胞激素組合中，IGF-1與IGF-2的組合濃度，瞭解IGF-1與IGF-2濃度調降後，腫瘤細胞球形成數量的變化……………………….......................... 45 圖3-6. 使用螢光細胞結合high content screening system分析IGF-1、IGF-2與IL-6組合的spheroid數量及面積……………………………………………………………………………........... 47 圖3-7. 加入STAT3與PI3K抑制劑對腫瘤細胞球的影響………………………………….......… 48 圖3-8. 給予IGF-1 10 ng/m+ IL-6 20 ng/mL細胞激素刺激後的STAT3、pSTAT3、Nanog蛋白質表現…………………………………………………………………………………………….. 49 圖3-9a-b. Sulf1毒性測試與Sulf1對細胞激素刺激腫瘤細胞球形成的影響……..........… 50 圖3-10. 透明質酸-幾丁質立體培養盤在不同塗層時所培養出的細胞形態…................. 52 圖3-11. 不同細胞濃度在不同濃度的HA修飾下，形成的Tumor spheroids…...........…. 53 圖3-12. 結合細胞激素與立體細胞培養盤的Sphere formation實驗……………….........… 56 表附錄 附表3-1. 將腫瘤相關纖維母細胞 (Cancer-associated fibroblasts, CAFs)與腫瘤幹細胞(Cancer stem cells, CSCs)及兩種細胞共培養後，有顯著變化的十六個基因的primer序列………………………………………………………………………………………………………….............. 57 | |
| dc.language.iso | zh-TW | |
| dc.subject | 腫瘤幹細胞 | zh_TW |
| dc.subject | 腫瘤相關纖維母細胞 | zh_TW |
| dc.subject | 腫瘤幹細胞 | zh_TW |
| dc.subject | 抗腫瘤幹細胞藥物快速篩選平台 | zh_TW |
| dc.subject | 無餵養細胞培養系統 | zh_TW |
| dc.subject | 抗腫瘤幹細胞藥物快速篩選平台 | zh_TW |
| dc.subject | 腫瘤微環境 | zh_TW |
| dc.subject | 腫瘤相關纖維母細胞 | zh_TW |
| dc.subject | 腫瘤微環境 | zh_TW |
| dc.subject | 無餵養細胞培養系統 | zh_TW |
| dc.subject | Cancer stem cell | en |
| dc.subject | Cancer-associated fibroblast | en |
| dc.subject | Microenvironment | en |
| dc.subject | 3D culture | en |
| dc.subject | IGF-1 | en |
| dc.subject | IL-6 | en |
| dc.subject | Cancer stem cell | en |
| dc.subject | Cancer-associated fibroblast | en |
| dc.subject | Microenvironment | en |
| dc.subject | 3D culture | en |
| dc.subject | IGF-1 | en |
| dc.subject | IL-6 | en |
| dc.title | 肺癌幹細胞株之無餵養細胞培養系統 | zh_TW |
| dc.title | Feeder Free Culture System For Lung Cancer Stem Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林泰元(Thai-Yen Ling),徐善慧(Shan-hui Hsu),陳健尉(Jan-Way Chen) | |
| dc.subject.keyword | 腫瘤幹細胞,腫瘤相關纖維母細胞,腫瘤微環境,抗腫瘤幹細胞藥物快速篩選平台,無餵養細胞培養系統, | zh_TW |
| dc.subject.keyword | Cancer stem cell,Cancer-associated fibroblast,Microenvironment,3D culture,IGF-1,IL-6, | en |
| dc.relation.page | 58 | |
| dc.identifier.doi | 10.6342/NTU201700641 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-02-16 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 毒理學研究所 | zh_TW |
| 顯示於系所單位： | 毒理學研究所 | |
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