RAF/MEK/ERK為一系列的訊號傳遞鏈，負責調控細胞的增生或凋亡。蕾莎瓦(Sorafenib)是一種多激酶抑制劑( multikinase inhibitor )，他被認為可以直接抑制RAF/MEK/ERK訊號傳遞鏈中的RAF，影響細胞存亡。蕾莎瓦在臨床上已經通過美國食品藥品監督管理局(Food and Drug Administration)許可，用來治療肝癌(advanced hepatocellular carcinoma)，但平均僅能延長病人2.8個月的壽命。 我們好奇治療效果如此有限的原因究竟為何，於是分析了許多肝癌細胞株，發現大部分的肝癌細胞對蕾莎瓦都是具有抗藥性的。RAF需要形成二聚體(dimer)才會活化下游的MEK，而蕾莎瓦濃度充足時能夠阻擋RAF二聚體上的兩個活化位，避免RAF的活化；但當蕾莎瓦的濃度不足，只阻擋了其中一個活化位時，反而會加速RAF二聚體的形成(dimerization)，加速傳遞鏈下游ERK的活化，將腫瘤微環境轉為不利於免疫細胞作用的狀態，導致治療出現抗藥性。
因此我們開發了一個能夠標靶肝癌細胞的奈米載體，同時包覆蕾莎瓦和MEK/ERK的抑制劑—AZD6244，可以減少RAF及ERK活化，減少癌細胞增生、調控PD-L1的表現、增加胞殺性T細胞( cytotoxic T cell )的浸潤，並且抑制血管新生，達到抑制腫瘤生長的目的。以標靶性奈米載體傳遞兩種抑制劑可以有效的降低蕾莎瓦造成的活化機制，克服治療肝癌時所面臨的抗藥性。

Sorafenib is a RAF inhibitor approved for several cancers, including hepatocellular carcinoma (HCC). However, sorafenib has limited efficacy in this disease. Inhibition of RAF kinases with anti-cancer drugs may induce a “paradoxical” upregulation of the downstream mitogen-activated protein kinase (MAPK) pathway, i.e., the RAF/MEK/ERK signal transduction cascade, both in cancer and normal cells. It is unknown whether “paradoxical” ERK activation occurs after sorafenib therapy in HCC, and has any role in treatment resistance. Here, we demonstrate that RAF inhibition by sorafenib rapidly leads to RAF dimerization and ERK activation in HCCs, which promotes treatment evasion. To overcome treatment evasion and reduce systemic effects, we developed CXCR4-targeted nanoparticles to co-deliver sorafenib with the MEK inhibitor AZD6244 in HCC. Using this approach, we preferentially and efficiently down-regulated RAF/ERK and PD-L1 expression in HCC, and facilitated intra-tumoral infiltration of cytotoxic CD8+ T cells. These effects resulted in a profound delay in tumor growth. Thus, this nano-delivery strategy to selectively target tumors and prevent the paradoxical ERK activation could increase the feasibility of dual RAF/MEK inhibition to overcome sorafenib treatment escape in HCC.

目錄
摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vii
縮寫 ix
第一章 緒論 1
1.1 肝癌 (Hepatocellular Carcinoma, HCC) 1
1.1.1 細胞增生與活化 2
1.1.2 細胞凋亡(Apoptosis) 4
1.1.3 血管新生 (Angiogenesis) 6
1.1.4 免疫逃脫反應 (Immune evasion) 8
1.2 蕾莎瓦 (Nexavar) 10
1.2.1 作用機制 10
1.2.2 RAF蛋白和蕾莎瓦作用位置與構型 12
1.2.3 副作用 14
1.2.3 抗藥性 15
1.3 奈米載體 16
1.3.1 聚乳酸聚乙醇酸Poly(lactic-co-glycolic acid), PLGA 19
1.3.2 標靶性配體 21
1.4 研究方向 23
第二章 實驗材料及方法 25
2.1 材料 25
2.2 細胞 25
2.3 奈米粒子(CTCE-NPs)製成 26
2.4 奈米粒子特性 29
2.5 藥物包覆率 29
2.6 細胞攝取 (cellular uptake) 29
2.7 受體競爭測試 (competition assay) 30
2.8 細胞存活率 (cell viability) 30
2.9 西方墨點法 30
2.10 動物模型 31
2.11 奈米粒子在小鼠體內組織分布(Biodistribution) 32
2.12 肝癌組織攝取奈米粒子 32
2.13 組織免疫染色 33
2.14 免疫沉澱法判斷BRAF-CRAF是否形成dimer 33
第三章 實驗結果 34
3.1 以蕾莎瓦治療出現的抗藥性 34
3.2 以Selumetinib克服蕾莎瓦的抗藥性 36
3.3 奈米粒子特性 38
3.4 CTCE-9908具備標靶肝癌的能力 39
3.6 肝癌細胞的存活率和MAPK pathway的調節有關，並且提高促細胞凋亡蛋白—BIM的表現 43
3.7 CXCR4靶向奈米載體在小鼠肝癌模型的功能 45
3.8 蕾莎瓦及Selumetinib共同作用可改善腫瘤微環境中免疫功能不全的現象 47
第四章 討論與結論 51
4.1討論 51
4.2結論 55
第五章 參考文獻 56

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