殼聚醣奈米纖維接枝玻尿酸以擴增並提高癌症幹細胞的比例

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洪振傑(Zhen-Jie Hong) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

殼聚醣奈米纖維接枝玻尿酸以擴增並提高癌症幹細胞的比例
(Proliferation and Enrichment of Cancer Stem Cells on Hyaluronic acid Grafted Chitosan Nanofibers)

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摘要(中)

殼聚醣是常用的支架材料，具有促進癌症細胞群聚成球狀並提升幹細胞的特性。為了可以模擬體內的微環境，因此我們利用電紡絲技術製備出殼聚醣奈米纖維絲，藉由奈米纖維的疊加形成3D孔洞結構。此外，為了進一步提升癌症幹細胞的選殖效果，我們將殼聚醣電紡絲以細胞外基質玻尿酸進行改質，並與殼聚醣膜做為對照。由生長曲線結果發現，玻尿酸改質的電紡絲因其近似腫瘤微環境(niche)環境，會使細胞生長數率略微減緩，但是相較於膜上的細胞，絲上的細胞生長速率有大幅的提升。而在抗藥性實驗結果證實，玻尿酸改質所篩選的細胞其化療的耐受度有大幅的提升。藉由即時聚合酶反應(qPCR)發現從改質的電紡絲上取下的細胞具有的細胞幹性、上皮間質轉換、基質降解能力、血管新生與抗藥性等基因表現均有提升。而這些結果也可以透過侵入性與細胞遷移實驗中得到驗證。最後我們利用流式細胞儀分析，發現與所篩選的細胞其癌症幹細胞相關的CD24及CD44表面標誌比例有大幅提升，證實我們所發展的殼聚醣奈米纖維接枝玻尿酸的確能提升癌症幹細胞的比例。此基材將所擴增篩選的細胞將有助於將來在抗癌藥物的開發和癌症治療的研究。

摘要(英)

Chitosan is a frequently used material for scaffold fabrication because it can promote cancer cells to form spheroid and enhance stem cell-relative characteristics. To mimic in vivo microenvironment, chitosan nanofibers were prepared using electrospinning technology, and their superposition can form a 3D porous structure. To effectively promote cancer stem cell adhesion, hyaluronic acid (HA) was grafted to electrospun chitosan fibers, and HA grafted chitosan films were applied as the control groups. The growth curve analysis demonstrated that HA modification reduced proliferation rates of surface cells because of its similarity to the cancer stem cell niche. On the other hand, cells grew on the fibers were faster than those on the films. The results of drug resistance examinations indicated that the chemotherapy tolerance of the cells collected from HA modified fibers was greatly improved. The levels of gene transcription determined by quantitative polymerase chain reaction (qPCR) indicated that the cells collected from HA modified fibers demonstrated high stemness, epithelial–mesenchymal transition, matrix degradation, angiogenesis, and drug resistance abilities. These results were consistent to their superior performances in colony formation, migration, and matrix invasion. Finally, flow cytometry analysis was applied to identify cell makers. The results indicated that the screened cells highly expressed the cancer stem cell-related makers CD24 and CD44, suggesting that the use of HA grafted chitosan fibers indeed increased the ratio of cancer stem cells. We expect these selected cells should be applicable to the development of anticancer drugs and the research of cancer therapy.

關鍵字(中)

★ 殼聚醣
★ 玻尿酸
★ 電紡絲
★ 癌症幹細胞

關鍵字(英)

論文目次

摘要 i
Abstract ii
致謝 iii
圖目錄 viii
表目錄 x
第一章緒論 1
1.1背景 1
1.2實驗目的 2
第二章文獻回顧 3
2.1癌症 3
2.1.1癌症幹細胞 3
2.1.2癌症幹細胞特性 4
2.1.3癌症幹細胞篩選 5
2.1.4癌症幹細胞相關基因 7
2.1.5癌症幹細胞表面標記 9
2.2電紡絲 11
2.2.1電紡絲原理 11
2.2.2電紡絲於幹細胞的應用 12
2.3玻尿酸 13
2.3.1玻尿酸之來源 13
2.3.2玻尿酸之性質 13
2.3.3玻尿酸於癌症幹細胞之應用 14
2.4殼聚醣 16
2.4.1殼聚醣之來源 16
2.4.2殼聚醣之性質 17
2.4.3殼聚醣於癌症幹細胞之應用 18
2.5電紡絲複合材料 19
2.5.1電紡絲複合材料之簡介 19
2.5.2殼聚醣接枝玻尿酸反應機制 20
2.5.3支架接枝玻尿酸於癌症幹細胞之應用 21
第三章實驗與方法 23
3.1實驗藥品 23
3.2實驗儀器 27
3.3實驗方法 29
3.3.1殼聚醣膜製備 29
3.3.2殼聚醣電紡絲纖維製備 29
3.3.3膜/纖維改質[68] 29
3.3.4纖維表面FT-IR分析 30
3.3.5纖維SEM樣本製備 30
3.3.6接觸角 31
3.3.7接枝率 31
3.3.8細胞培養 32
3.3.9細胞培養於膜和絲上型態變化 33
3.3.10細胞培養於膜和絲上細胞球大小及個數統計 33
3.3.11生長曲線實驗 34
3.3.12抗藥性實驗 34
3.3.13 Alamar Blue分析 34
3.3.14即時聚合酶反應酶(qPCR) 35
3.3.15細胞遷移實驗[33] 39
3.3.16侵入性實驗[84] 39
3.3.17流式細胞分析儀 40
第四章結果與討論 42
4.1電紡絲纖維改質後傅立葉紅外線FTIR-ATR分析 42
4.2電紡絲纖維改質後SEM分析 43
4.3比較殼聚醣膜與殼聚醣電紡絲 45
4.3.1接觸角 45
4.3.2接枝率 47
4.3.3細胞型態 48
4.3.4細胞球大小及個數 52
4.3.5生長曲線 55
4.3.6抗藥性 57
4.4癌症幹細胞相關基因表現 60
4.4.1幹性相關基因 60
4.4.2上皮間質轉換相關基因 62
4.4.3侵入性相關基因 64
4.4.4血管新生相關基因 65
4.4.5抗藥性相關基因 66
4.5癌症幹細胞之細胞遷移 67
4.6癌症幹細胞之侵入能力 69
4.7流式細胞術 71
第五章結論 73
第六章參考文獻 75

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指導教授

胡威文(Wei-Wen Hu)

審核日期

2017-8-23

推文