臺灣博碩士論文加值系統

黑色素癌是一種好發於皮膚的惡性腫瘤，容易發生淋巴轉移以及血液轉移，轉移至肺部、腦部、消化道等器官而導致高致死率。臨床治療原位性黑色素癌主要是手術切除輔以化學療法或放射治療，針對遠期的黑色素轉移以姑息性的化學療法為主，而近期發展的免疫療法則還在研究中，主要原因在於黑色素癌對於化學療法以及放射治療都具有相當高的抗性，且容易發生轉移的現象，加上無法預測其轉移的時間，導致黑色素癌難以被治療。因此，預測黑色素癌轉移的情況以及時間點有其重要性，先前的文獻指出在腫瘤預轉移的肺部有嗜中性球(Neutrophils)大量浸潤的現象，因此，血液中的髓質免疫抑制細胞(MDSCs)或許可以當做一個黑色素癌轉移的指標。另一方面，在腫瘤轉移過後，化學療法對於髓質免疫抑制細胞又會產生什麼影響。
本研究中，以黑色素細胞(B16_F0)作為研究的模式，應用化療藥物Cisplatin探討在黑色素癌轉移過程中髓質免疫抑制細胞(MDSCs)的變化及化療藥物治療後的變化與療效的關係。
結果顯示，在尾靜脈注射黑色素癌之後，第十六天發生明顯肺轉移，而在腫瘤發生轉移的過程中，透過流式細胞儀(Flowcytometry)分析髓質免疫抑制細胞中處於發炎反應的單核球細胞(inflammatory -monocytes)以及嗜中性球(Neutrophils)兩個細胞族群，分別在第八天以及第十五天有上升的趨勢以及明顯的峰值，可以做為肺轉移發生的指標。而經過化學療法過後，嗜中性球呈現上升的趨勢，處於發炎反應的單核球則呈現相反的趨勢，而肺轉移的病灶明顯減少，但卻產生肝轉移的現象。這些結果顯示，髓質免疫抑制細胞的確可以當做預測黑色素癌轉移的指標，而經過化學療法過後造成黑色素癌發生肝轉移，可能與嗜中性球與處於發炎反應的單核球有關係，但相關機制尚未明確。

Melanoma is malignant tumor that occurs in the skin and frequently metastasis via lymph node and blood into lung, brain, gastrointestinal tract and other organs that leads to high mortality rate. Melanoma is mainly treated by surgical resection combined with chemotherapy or radiation therapy for tumor in situ. If metastasis occurs, palliative chemotherapy is frequently used. Many immunotherapy protocols for melanoma are currently under trials. Melanoma is hard to be cured because of the high resistance to irradiation and chemo-drugs and high frequency of metastasis to risked organs. A protocol that can forecast the treatment response and state of metastasis will be valuable for developing strategy against melanoma. This study aimed to examine if MDSCs in blood could be an index to predict metastasis and the response to chemotherapy. Using B16-F0 as metastasis model, the variability of MDSCs in the blood during the progression of metastasis and following chemotherapy was examined by flow cytometry. Following i.v. injection of B16 tumor cells, the number of inflammatory monocytes has reached its highest level at day 8 and the neutrophils had reached its highest level at day 15, one day prior to detectable lung metastatic foci. The administration of ciplatin did not alter the pattern of neutrophils ,but reduced the number of inflammatory monocytes. Although B16 tumor cells is sensitive to the cytotoxicity of cisplatin in vitro, the administration of cisplatin did not significantly improve the survival. Histology shows that mice receiving cisplatin treatment mainly died from liver metastasis, instead of lung metastasis in control mice. In summary, this study demonstrates that cisplatin treatment could alter the path of metastasis of melanoma and could be associated with the alternation of sub-population of MDSCs. The MDSCs in the blood is a potential index to predict the status of melanoma metastasis for designing new combination treatment protocol.

目錄
英文摘要 I
中文摘要 II
致謝 III
目錄 IV
第一章 序論 1
1.1黑色素瘤 1
1.1.1黑色素癌簡介 1
1.1.2 黑色素癌的診斷 1
1.1.3黑色素癌的治療 2
1.1.4 黑色素癌治療面臨的挑戰 3
1.2化學療法 3
1.2.1 化學療法 3
1.2.2 順鉑(Ciaplatin) 4
1.3髓質免疫抑制細胞(MDSCs) 4
1.3.1 髓質免疫抑制細胞的特性 4
1.4實驗目的與內容 5
第二章 材料與方法 7
2.1細胞培養 7
2.1.1 配製DMEM細胞培養液 7
2.1.1 細胞培養液的配方 7
2.1.2 細胞繼代 8
2.1.3 黑色素癌細胞對於Cisplatin的敏感度試驗 8
2.1.4 細胞存活試驗(MTT assay) 9
2.2動物實驗 10
2.2.1 動物來源 10
2.2.2 動物分組 10
2.2.3 植入B16-F0腫瘤細胞株 10
2.2.4 注射Cisplatin 10
2.2.5 老鼠存活分率 11
2.2.6 血液樣本處理 11
2.2.7 抗體處理 12
2.2.8 流式細胞儀上機 13
2.2.9 腫瘤轉移以及治療效果評估 14
第三章 實驗結果 15
3.1細胞實驗 - in vitro 15
3.1.1黑色素癌細胞對於Cisplatin的敏感度試驗 15
3.2動物實驗 - in vivo 16
3.2.1黑色素癌轉移 16
3.2.2小鼠存活曲線 16
3.2.3小鼠血液中CD11b+細胞比例變化 17
3.2.4小鼠血液中嗜中性球細胞比例變化 18
3.2.5小鼠血液中單核球比例變化 18
3.2.6小鼠血液中異質性髓質細胞比例變化 19
3.2.7小鼠血液中發炎反應之單核球比例變化 20
3.2.8經過Cisplatin治療後的肺轉移 21
3.2.9經過Cisplatin治療後的肝轉移 21
第四章 討論 23
【圖表說明】 29
【圖表】 32
【參考文獻】 42

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