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

【參考文獻】
1. Group, U.C.S.W., United States Cancer Statistics: 1999–2011 incidence and mortality web-based report. Atlanta (GA): Department of Health and Human Services, Centers for Disease Control and Prevention, and National Cancer Institute, 2014.
2. Jemal, A., et al., Global cancer statistics. CA: a cancer journal for clinicians, 2011. 61(2): p. 69-90.
3. Rigel, D.S., J. Russak, and R. Friedman, The evolution of melanoma diagnosis: 25 years beyond the ABCDs. CA: a cancer journal for clinicians, 2010. 60(5): p. 301-316.
4. Gray-Schopfer, V., C. Wellbrock, and R. Marais, Melanoma biology and new targeted therapy. Nature, 2007. 445(7130): p. 851-857.
5. Garrison, M. and L. Nathanson. Prognosis and staging in melanoma. in Seminars in oncology. 1996.
6. Balch, C.M., et al., Final version of 2009 AJCC melanoma staging and classification. Journal of Clinical Oncology, 2009. 27(36): p. 6199-6206.
7. Avril, M., et al., Regression of primary melanoma with metastases. Cancer, 1992. 69(6): p. 1377-1381.
8. Sober, A.J. and C.M. Balch, Method of biopsy and incidence of positive margins in primary melanoma. Annals of surgical oncology, 2007. 14(2): p. 274-275.
9. Harland, C., et al., Differentiation of common benign pigmented skin lesions from melanoma by high‐resolution ultrasound. British Journal of Dermatology, 2000. 143(2): p. 281-289.
10. Solis, O.J., et al., Intracerebral metastatic melanoma: CT evaluation. Computerized tomography, 1977. 1(2): p. 135-143.
11. Pennasilico, G., et al., Magnetic resonance imaging in the diagnosis of melanoma: in vivo preliminary studies with dynamic contrast-enhanced subtraction. Melanoma research, 2002. 12(4): p. 365-371.
12. Miao, Y., et al., 203Pb-Labeled α-Melanocyte–Stimulating Hormone Peptide as an Imaging Probe for Melanoma Detection. Journal of Nuclear Medicine, 2008. 49(5): p. 823-829.
13. Miao, Y., K. Benwell, and T.P. Quinn, 99mTc-and 111In-labeled α-melanocyte-stimulating hormone peptides as imaging probes for primary and pulmonary metastatic melanoma detection. Journal of Nuclear Medicine, 2007. 48(1): p. 73-80.
14. Doss, L., Radiation therapy in malignant melanoma. Missouri medicine, 1979. 76(12): p. 641.
15. Bajetta, E., et al. Metastatic melanoma: chemotherapy. in Seminars in oncology. 2002. Elsevier.
16. Mackensen, A., et al., Phase I study of adoptive T-cell therapy using antigen-specific CD8+ T cells for the treatment of patients with metastatic melanoma. Journal of Clinical Oncology, 2006. 24(31): p. 5060-5069.
17. Morton, D., et al., Immunological factors which influence response to immunotherapy in malignant melanoma. Surgery, 1970. 68(1): p. 158-63; discussion 163-4.
18. Eigentler, T.K., et al., Palliative therapy of disseminated malignant melanoma: a systematic review of 41 randomised clinical trials. The lancet oncology, 2003. 4(12): p. 748-759.
19. Johnson, T.M., et al., Advances in melanoma therapy. Journal of the American Academy of Dermatology, 1998. 38(5): p. 731-741.
20. Overgaard, J., The role of radiotherapy in recurrent and metastatic malignant melanoma: a clinical radiobiological study. International Journal of Radiation Oncology* Biology* Physics, 1986. 12(6): p. 867-872.
21. Albert, D.M., A.S. Niffenegger, and J.K. Willson, Treatment of metastatic uveal melanoma: review and recommendations. Survey of ophthalmology, 1992. 36(6): p. 429-438.
22. Rosenberg, S.A., et al., Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report. The New England journal of medicine, 1988. 319(25): p. 1676-1680.
23. Parkinson, D.R., et al., Interleukin-2 therapy in patients with metastatic malignant melanoma: a phase II study. Journal of Clinical Oncology, 1990. 8(10): p. 1650-1656.
24. Yuan, J., et al., CTLA-4 blockade enhances polyfunctional NY-ESO-1 specific T cell responses in metastatic melanoma patients with clinical benefit. Proceedings of the National Academy of Sciences, 2008. 105(51): p. 20410-20415.
25. Barranco, S., M. Romsdahl, and R. Humphrey, The radiation response of human malignant melanoma cells grown in vitro. Cancer research, 1971. 31(6): p. 830-833.
26. Röckmann, H. and D. Schadendorf, Drug resistance in human melanoma: mechanisms and therapeutic opportunities. Oncology Research and Treatment, 2003. 26(6): p. 581-587.
27. Tattersall, M., Cancer Chemotherapy—Fundamental Concepts and Recent Advances. Journal of clinical pathology, 1976. 29(4): p. 367.
28. Coates, A., et al., On the receiving end—patient perception of the side-effects of cancer chemotherapy. European Journal of Cancer and Clinical Oncology, 1983. 19(2): p. 203-208.
29. Ozols, R.F., et al., Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a Gynecologic Oncology Group study. Journal of Clinical Oncology, 2003. 21(17): p. 3194-3200.
30. Schiller, J.H., et al., Comparison of four chemotherapy regimens for advanced non–small-cell lung cancer. New England Journal of Medicine, 2002. 346(2): p. 92-98.
31. Roberts, J., The mechanism of action of anti-tumor platinum compounds. Prog. Nucleic. Acid Res. Mol. Biol., 1979. 22: p. 71-133.
32. Sorenson, C.M. and A. Eastman, Influence of cis-diamminedichloroplatinum (II) on DNA synthesis and cell cycle progression in excision repair proficient and deficient Chinese hamster ovary cells. Cancer Research, 1988. 48(23): p. 6703-6707.
33. Barry, M.A., C.A. Behnke, and A. Eastman, Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochemical pharmacology, 1990. 40(10): p. 2353-2362.
34. Florea, A.-M. and D. Büsselberg, Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers, 2011. 3(1): p. 1351-1371.
35. Arany, I. and R.L. Safirstein. Cisplatin nephrotoxicity. in Seminars in nephrology. 2003. Elsevier.
36. Schweitzer, V.G., Cisplatin-induced ototoxicity: the effect of pigmentation and inhibitory agents. The Laryngoscope, 1993. 103(4 Pt 2): p. 1-52.
37. Nematbakhsh, M., et al., A histopathological study of nephrotoxicity, hepatoxicity or testicular toxicity: Which one is the first observation as side effect of Cisplatin-induced toxicity in animal model? Journal of nephropathology, 2012. 1(3): p. 190.
38. Singh, U.P., et al., Role of resveratrol-induced CD11b+ Gr-1+ myeloid derived suppressor cells (MDSCs) in the reduction of CXCR3+ T cells and amelioration of chronic colitis in IL-10−/− mice. Brain, behavior, and immunity, 2012. 26(1): p. 72-82.
39. Gabrilovich, D.I. and S. Nagaraj, Myeloid-derived suppressor cells as regulators of the immune system. Nature Reviews Immunology, 2009. 9(3): p. 162-174.
40. Peranzoni, E., et al., Myeloid-derived suppressor cell heterogeneity and subset definition. Current opinion in immunology, 2010. 22(2): p. 238-244.
41. Nagaraj, S., et al., Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nature medicine, 2007. 13(7): p. 828-835.
42. Corzo, C.A., et al., HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment. The Journal of experimental medicine, 2010. 207(11): p. 2439-2453.
43. Highfill, S.L., et al., Bone marrow myeloid-derived suppressor cells (MDSCs) inhibit graft-versus-host disease (GVHD) via an arginase-1–dependent mechanism that is up-regulated by interleukin-13. Blood, 2010. 116(25): p. 5738-5747.
44. Filipazzi, P., et al., Identification of a new subset of myeloid suppressor cells in peripheral blood of melanoma patients with modulation by a granulocyte-macrophage colony-stimulation factor–based antitumor vaccine. Journal of Clinical Oncology, 2007. 25(18): p. 2546-2553.
45. Zea, A.H., et al., Arginase-producing myeloid suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion. Cancer research, 2005. 65(8): p. 3044-3048.
46. Granot, Z., et al., Tumor entrained neutrophils inhibit seeding in the premetastatic lung. Cancer Cell, 2011. 20(3): p. 300-14.