大腸直腸癌的發生率及死亡率在全球一直都排名在前，多因於手術後復發並且轉移至其他器官，導致預後往往不佳，終至病人死亡。過去對於癌症的研究不在話下，但效果始終因為癌症幹細胞的產生，造成侵襲力提升、抗藥特性等而打折扣，然而近十幾年來，大家陸陸續續把焦點拉出針對癌細胞本身，而開始著手研究腫瘤周遭的微環境(Microenvironment)，發現腫瘤微環境在多種癌症中做為引導腫瘤發展或惡化轉移的調控者。大腸直腸癌微環境中，癌相關纖維母細胞(Carcinoma associated fibroblast, CAF) 為主要的組成成員，其在癌變的進展過程中扮演著重要角色，主要基於CAFs為主要分泌趨化因子的來源細胞之一。第一基質衍生因子(stromal cell-derived factor 1, SDF-1)，亦稱CXCL12，是一種趨化因子(chemokine)，目前已有許多研究指出在乳癌及肺癌中 SDF-1主要由纖維母細胞所分泌，並與促進癌細胞生長及血管新生有關。然而SDF-1在大腸直腸癌細胞中的角色以及其分子機制都尚未被釐清。
我們發現CAFs中SDF-1及其接受器CXCR4的表現與在NFs中相比均有顯著的上升，於是我們進一步分析SDF-1對大腸直腸癌的侵襲轉移能力是否有影響。利用重組SDF-1蛋白(模擬CAFs旁分泌出的SDF-1)處理細胞後，發現SDF-1可以刺激癌細胞進行上皮-間葉轉型現象並促進侵襲轉移；同時強化癌症幹細胞的特性。初步驗證腫瘤微環境中CAFs分泌之SDF-1會經由旁分泌作用影響大腸直腸癌之侵襲轉移及癌症幹細胞特性後，我們進一步分析當癌細胞受到SDF-1刺激後，能否經由本身自分泌作用增加癌細胞的惡性度。透過我們所建立的SDF-1過表現之大腸直腸癌細胞株發現，SDF-1過表現之細胞株會出現顯著上皮-間葉轉型現象並促進侵襲轉移；同時，也會增加幹細胞特性及抗藥性，進而增加癌細胞的惡性度。由我們的結果顯示SDF-1會透過旁分泌及自分泌作用，雙管齊下增加癌細胞惡性度。動物實驗結果也顯示SDF-1會刺激腫瘤生長，並表現顯著的癌幹細胞標誌。未來值得我們深入研究自分泌透過的訊號傳遞路徑分子機制與旁分泌的差異，藉由兩者路徑的釐清，可以有效調控SDF-1的生成，並希望改善大腸直腸癌的預後。
這些結果釐清了CAFs分泌的SDF-1在大腸直腸癌進行過程中扮演之角色，藉本論文的完成將可明瞭大腸癌細胞侵襲、轉移過程中，癌細胞與微環境中CAFs間互動之相關分子機制，進而建立一個以SDF-1為標靶的創新治療策略。

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. The prognosis of CRC is usually poor because of its propensity for extensive invasion, local recurrence and frequent regional lymph node metastasis. Researches have shown Carcinoma-associated fibroblasts (CAFs), a major type of tumor-surrounding stromal cell, generate mediators, such as Stromal Cell-Derived factor 1 (SDF-1), through which they interact with tumors and contribute to the progression and increase of stemness of cancer. The orchestration between CAFs and CRC cells is complex. Despite recent studies demonstrating the paracrine effect of stromal cells in the tumor microenvironment on initiation and progression of colorectal cancer cells, the major mediator related to CAFs and its underlying mechanism still remain unknown.
Based on our present study, we found that the expression of SDF-1 and its receptor C-X-C chemo receptor type 4 (CXCR4) are stronger when co-cultured with CAFs than with NFs. In addition, ELISA assay also validates the presence of SDF-1 that is responsible for the crosstalk between fibroblasts and CRC cells via the paracrine effect. Furthermore, the mediator SDF-1 not only triggers epithelial-mesenchymal-transition (EMT), showing upregulation of EMT markers in RNA and proteins levels, but also improves the capabilities of migration and invasion. CRC cells treated with recombinant SDF-1, mimics the CAFs-CRC paracrine route, increased capabilities of sphere formation in ten days. To simulate SDF-1-induced autocrine signaling, we established stable clones of SDF-1-overexpressing CRC cells. Stable clone CRC cells exhibited the epithelial-mesenchymal-transition, increased mobility and upregulation of drug-resistant genes. In vivo study reconfirmed the functional role of SDF-1 in tumor initiation. Immunohistochemistry of tumor dissected from mouse confirmed that autocrined-SDF-1 enhances CSCs properties through different pathways than paracrine. Besides the paracrine signaling, we clearly verified that CAFs-induced SDF-1 reciprocally triggered cancer cells to produce SDF-1 in an autocrine manner, which resulted in the binding to CXCR4 and cancer progression. Further studies include evaluations of the mechanism of autocrine signaling. Meanwhile, validates in clinical patients.
Our results verify that CAFs promote cancer invasiveness via paracrine and autocrine effects on microenvironmental SDF-1 signaling, and suggest that SDF-1 is a potentially biomarker which contributes to the expression of CSC properties, and may provide as a therapeutic option for improving prognosis in patients with CRC.

謝誌 i
目錄 iii
圖次目錄 iv
表次目錄 v
英文縮寫 vi
中文摘要 vii
Abstract ix
壹‧序論 1
貳‧研究方向與目的 6
參‧材料與方法 8
肆‧實驗結果 24
伍‧討論 33
陸‧結論 38
柒‧參考文獻 41
捌‧附圖 45
玖‧附表 62
拾‧附錄 65

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