本研究將以生物醫學工程的角度使用明膠以及羥磷灰石去仿製出接近真實人骨結構的生物材料支架，在其中放入高度侵略性的乳腺癌細胞，結合使用在許多乳癌研究中提出可活化乳癌細胞的細胞因子，以建立乳癌發生骨轉移後的實驗仿體，並藉此仿體探究骨轉移微環境。實驗起始將先探討表皮生長因子對於乳腺癌細胞之影響，再逐步將實驗流程轉移到生物材料上，最終再將此模組導入三維的仿骨生物材料支架，並嘗試討論骨轉移仿體的腫瘤組織型態以及表皮生長因子在微觀三維結構下對乳癌細胞狀態的影響。
實驗結果可以得知，由羥磷灰石以及明膠所製成的仿骨支架可以形成大小約略100-500 μm左右的多微孔架構，並可在仿骨支架中培養乳腺癌細胞後形成乳癌骨轉移仿體。仿體經組織切片可呈現與真實的骨轉移組織相似結構，具有各式孔洞以及孔徑大小各異的網狀結構且含有乳腺癌細胞在其中。更可以非侵入之螢光標記方式檢測乳腺癌細胞在仿體內之位置以及數量差別。最後在微觀立體環境的觀測下，可見乳腺癌細胞會因環境的構成成分與結構不同，出現形態各異的細胞形態以及生長環境的偏好，如：癌細胞偏好在羥磷灰石表面生長而非明膠表面以及癌細胞可順應支架立體的表面結構進行貼附生長等。而表皮生長因子更可以改變癌細胞在這些環境中既定的形態並且促進增生。

In this study, we engineered bone-like biomaterial scaffolds by gelatin and hydroxyapatite, cultured highly invasive breast cancer into scaffolds. We chose a kind of cytokine as breast cancer activator, epidermal growth factor (EGF), which is often mention in others breast cancer relative studies. In order to imitate the pro-metastatic bone microenvironment, we tested the effects of EGF to breast cancer cells in traditional way, then shifted the experiment ideal to scaffolds. Finally, we built bone metastasis models to discuss breast cancer and the effects of EGF in 3D microenvironment.
Gelatin-hydroxyapatite scaffolds can provide 3D platform for breast cancer cells growth and formed bone metastasis phantoms, which porous size are emerged from 100 to 500 μm. In the results of tissue section, we can get bone metastasis-like results, contained variety of porous size, structure and breast cancer cell inside it. Additionally, we can detect the breast cancer location and amount in scaffolds by non-invasive fluorescent detection. Finally, we discuss the results in phantom microenvironment. Depending on scaffolds’ structure and composition, results show that cancer cells might change morphology and choose the growing surface. For example, cancer cells prefer to grow on hydroxyapatite surface than gelatin surface, and the growth can match to scaffolds surface. Moreover, EGF might change these facts, such as promote cancer cells’ proliferation and change their morphology in improper surface, like gelatin.

中文摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VIII
表目錄 X
第1章 緒論 1
1.1 研究動機 1
1.2 現今概況 2
1.2.1 活體外骨轉移研究 2
1.2.2 活體骨轉移研究 3
1.2.3 臨床上骨轉移研究 4
1.3 研究目的 4
第2章 理論基礎 6
2.1 乳癌骨轉移 6
2.1.1 乳癌 6
2.1.2 骨轉移 7
2.2 腫瘤微環境 9
2.2.1 腫瘤微環境中的細胞外基質 9
2.2.2 表皮生長因子 12
2.2.3 表皮生長因子對乳癌細胞影響 12
2.3 腫瘤仿體 14
2.3.1 組織仿體材料 14
2.3.2 仿體培養 17
2.4 腫瘤觀察方式 18
2.4.1 非侵入式觀察 18
2.4.2 侵入式觀察 19
第3章 材料與方法 22
3.1 實驗藥品 22
3.2 實驗儀器 23
3.3 實驗流程圖 24
3.4 乳癌骨轉移仿體 25
3.4.1 仿骨支架 25
3.4.1.1 支架製備 25
3.4.1.2 孔洞率分析 29
3.4.1.3 製備骨轉移微環境 31
3.4.2 乳癌細胞 34
3.4.2.1 乳癌細胞選擇與來源 34
3.4.2.2 乳癌細胞繼代 35
3.4.2.3 乳癌細胞活性測試 36
3.4.3 乳癌與仿骨支架共培養 39
3.4.3.1 生物反應器 39
3.4.3.2 動態培養 41
3.5 乳癌骨轉移仿體分析 42
3.5.1 非侵入式層面 42
3.5.2 侵入式層面 42
3.5.2.1 病理切片觀測 42
3.5.2.2 微觀骨轉移環境觀測 43
3.5.2.3 微觀骨轉移環境觀中細胞偽足分析 43
第4章 結果與討論 44
4.1 三維仿骨支架之特性 44
4.1.1 支架結構觀察與討論 44
4.1.2 支架孔洞調控與分析 47
4.1.2.1 支架液體滲透性 47
4.1.2.2 支架孔洞大小調控 48
4.1.2.3 支架孔洞率分析 49
4.2 表皮生長因子對乳癌細胞之作用效果 51
4.2.1 對乳癌細胞增生之影響 51
4.2.2 對乳癌細胞形態之影響 55
4.2.3 對乳癌細胞遷移之影響 56
4.2.4 本章小結與討論 61
4.3 乳癌細胞在仿骨微環境之分析 62
4.3.1 仿體組織層面分析 62
4.3.1.1 切片分析 62
4.3.1.2 IVIS量化分析 67
4.3.2 仿體微觀分析 72
4.3.2.1 仿骨支架表面狀態與細胞間的關係 72
4.3.2.2 表皮生長因子在仿骨支架對細胞形態影響 76
4.3.2.3 表皮生長因子對乳腺癌細胞仿體組織形成之影響 82
第5章 結論 85
第6章 參考資料 87
第7章 附錄 92
7.1 參與研討會與發表 92

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