探討黑色素瘤條件培養基對於繼發性腫瘤的產生之研究

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姓名

林哲宇(Che-Yu Lin) 查詢紙本館藏

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系統生物與生物資訊研究所

論文名稱

探討黑色素瘤條件培養基對於繼發性腫瘤的產生之研究
(Study of the conditioned medium from the melanoma cells in secondary tumor formation)

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至系統瀏覽論文 (2027-10-19以後開放)

摘要(中)

Vemurafenib (PLX4032) 是一種小分子的抑制劑用來治療由 BRAF 基因突變 (BRAFV600E)的黑色素瘤。在臨床上發現到黑色素瘤患者使用 BRAFV600E抑制劑治療後會產生抗藥性以及皮膚相關的不良反應發生，在這些不良反應事件當中包括繼發性腫瘤的產生，目前 BRAFV600E 抑制劑誘導繼發性腫瘤發展的機制尚未完全確定。有文獻提到，在不良反應當中所產生的繼發性腫瘤有 21%-60% 是 RAS 突變，這增強了 BRAF-CRAF 異二聚體的形成，從而激活了 MEK/ERK 通路。我們假設在腫瘤微環境當中，細胞之間的溝通會傳遞某些因子而造成影響有，可能是經由 BRAFV600E 抑制劑治療黑色素瘤細胞所釋放的因子並且有助於正常角質形成細胞和表皮細胞形成繼發性腫瘤。在這項研究中，我們利用BRAFV600E 抑制劑 PLX4032 或 PLX8394（一種新型 BRAFV600E 抑制劑）處理黑色素瘤細胞收集的條件培養基 (CM)來驗證我們研究的假設，看是否條件培養基當中存在某些因子而誘導繼發性腫瘤的產生。通過傷口癒合和軟瓊脂測定法來檢測使用 CM 培養的表皮細胞的遷移和不依賴性貼壁的生長。結果表明，用黑色素瘤細胞的 CM 培養正常角質形成細胞和上皮細胞將顯著增強它們的遷移和不依賴性貼壁的生長。在腫瘤微環境中由於細胞外囊泡 (EVs)可能作為在細胞間溝通的重要介質，因此我們分離出黑色素瘤細胞所釋放的 EVs 觀察是否會影響角質細胞的遷移。在實驗中，我們從條件培養基中分離 EVs，利用西方墨點法與免疫標記後經由穿透式電子顯微鏡觀察其結構與特性，確認純化出的 EVs 綜合上述結論我們猜測，用黑色素瘤細胞所釋放的 EVs 可能會促進角質細胞或上皮細胞的腫瘤發生。

摘要(英)

Vemurafenib (PLX4032) is a small molecule inhibitor of the mutant form of BRAF gene (BRAFV600E) for the treatment of melanoma. The resistance of BRAF inhibitor produced the secondary tumors in patients. Until now, the mechanisms of BRAFV600E inhibitor-induced secondary tumor development is not fully defined. It has been reported that most of the second tumors have 21%-60% RAS mutations, which enhances the BRAF-CRAF heterodimer formation to activate the MEK/ERK pathway. However, we hypothesize that the intercellular communication especially factors released from melanoma treated with BRAFV600E inhibitors may contribute to the
formation of secondary tumors which are originated from normal keratinocytes and epidermal cells. In this study, we utilized the conditioned medium (CM) collected from melanoma cells treated with BRAFV600E inhibitor, PLX4032, or PLX8394, a novel BRAFV600E inhibitor, to investigate our hypothesis. The migrative, and tumorigenic abilities of epidermal cells cultured with CM were detected by wound healing, and soft
agar assays. The results showed that normal keratinocytes and epithelial cells cultured with CM from melanoma cells treated with PLX4032 would enhance their migration,
and anchorage-independent growth as compared to the control group significantly. Since extracellular vehicles (EVs) as the important mediators that may act as cell-tocell communication in the tumor microenvironment, we investigated whether the EVs released from melanoma can affect tumorigenesis of keratinocytes or epithelial cells. Therefore, we isolated EVs from the conditioned medium, and confirmed the structure and characterization by Western blotting and TEM image. Taken together, our results
suggest that melanoma cells may release EVs to promote tumorigenesis in keratinocytes or the normal epithelial cells.

關鍵字(中)

★ 黑色素瘤
★ 條件培養基

關鍵字(英)

★ melanoma
★ conditioned medium

論文目次

探討黑色素瘤條件培養基對於繼發性腫瘤的產生之研究摘要 i
Abstract ii
致謝 iii
圖目錄 vii
Abbreviation list viii
一、介紹Introduction 1
1.黑色素瘤 (Melanoma) 1
1-1皮膚癌 (Skin cancer) 1
1-2.黑色素瘤的基因突變 (the mutations of melanoma) 2
1-3黑色素瘤的治療 (the treatment of melanoma) 3
2.BRAF抑制劑 4
2-1 BRAF抑制劑所影響的機制 (Mechanisms of BRAF Inhibitors) 4
2-2 BRAF抑制劑所造成的副作用 (Side Effects of BRAF Inhibitors) 4
2-3繼發性腫瘤的機制 (Mechanisms of Secondary Tumors) 5
3.細胞訊息傳遞 (Signal transduction) 5
3-1細胞間的溝通 5
3-2腫瘤微環境 (The tumor microenvironment) 6
4.研究目的及動機 7
二、實驗材料與方法 8
1.實驗材料（Material）： 8
1.1細胞株（Cell lines） 8
1.2藥物（Drugs） 8
1.3抗體（Antibodies） 8
2實驗方法（Methods）： 8
2-1蛋白質提取物的製備（Preparation of protein extraction） 8
2-2西方墨點法（Western Blot analysis） 9
2-3條件培養基製備（Preparation of conditioned medium） 9
2-4製備耗盡外泌體的培養基 (Preparation of EV-Depleted BSA Culture Medium) 10
2-5細胞不依賴性生長 (Anchorage-independent growth) 10
2-6細胞生長偵測實驗（Cell proliferation assay） 10
2-7細胞爬行實驗 (Cell migration assay) 10
2-8細胞外泌體的純化及分析 (Isolation and analysis of EVs） 11
2-8-1細胞外泌體的蛋白質分析（Analysis proteins of EVs） 11
2-8-2免疫金標記（Immunogold labeling） 11
2-9統計（Statistics） 12
三、實驗結果 13
1. 用黑素瘤細胞條件培養基誘導表皮細胞不依賴貼壁生長。 13
2. 用黑素瘤細胞條件培養基誘導角質細胞不依賴貼壁生長。 13
3. 用黑素瘤細胞條件培養基誘導角質細胞的爬行能力增加。 14
4. 從黑色素瘤的條件培養基中分離出外泌體。 16
5. 從黑色素瘤分泌出的外泌體對角質細胞爬行能力的影響。 16
四、結論 18
五、討論 19
1. 角質細胞經由PLX4032治療的條件培養基對於野生型BRAF的影響。 19
2. 黑色素瘤經由PLX8394 與PLX4032治療的條件培養基對於表皮細胞的能力影響。 19
3. 黑色素癌細胞所分泌出來的外泌體是否會影響表皮細胞。 19
4. 黑色素癌細胞經由PLX4032的治療所釋放的外泌體是否有差異。 20
5. 外泌體的應用及發展 20
六、參考文獻 22

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指導教授

馬念涵((Nian-Han Ma)

審核日期

2022-10-20

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