李明亭臺灣大學：生化科學研究所高文德Kao, Ven-TheVen-TheKao2007-11-262018-07-062007-11-262018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/52757稍早的文獻顯示，並非所有的癌細胞都具有轉移的能力，真正具備轉 移能力的癌細胞可能只佔整個族群的0.1%，如何篩選出具有轉移能力的癌 細胞，成為近幾年重要的課題。癌細胞入侵血管是癌細胞轉移過程中最為 關鍵的步驟，本實驗室利用in vitro invasion assay，從A431 P 成功的 篩選出一批具有較強入侵能力的細胞，命名為A431 III，隨後進行細胞特 性之分析，並尋求可能的分子機制以解釋為何A431 III 比A431 P 更具侵 略性。 癌細胞要入侵，必須具備：１）降低細胞間附著力，２)增強細胞之 移動能力，３）增加matrix metalloproteinase (MMP)的釋放，分解 extracellular matrix (ECM)，以利細胞之移動。初步的檢測顯示，A431 III 提高了MMP-9 之分泌量，並且具備較強的移動能力。此外，於cell spreading assay，我們發現A431 III 具有較強之細胞延展性，能過度活 化下游FAK，造成FAK 磷酸化上升。這些結果顯示A431 III 提升細胞之移 動能力與MMP 之分泌，可能經由FAK 調控，最終導致細胞具入侵性。It was reported previously that not all of primary tumor possesses metastatic ability, and only about 0.1 percent tumor cells display higher invasive potential. How to isolate and identify these high invasive cells from primary tumor cells became an important issue in this field. Cell invasion is a fundamental component of tumor cell metastasis. In this study we use in vitro invasion assay appartus to select higher invasive carcinoma cells. The assay was performed by A431 P tumor cells on a porous membrane coated with matrix, after which the cell invading the matrix were collected (designated A431 I) and subcultured. The same assay was repeated until the A431 III was obtained. We then explore whether the A431 III has higher invasive potential. First, using gelatin zymography, we observed that A431 III secreted higher amounts of MMP-9 than that of A431 P. Secondly, using wound healing assay, cell attachment and spreading experiment. We observed that A431 III exhibit higher motility potential as well. This data suggest that A431 III possesses higher metastatic potential and FAK might play important role in regulate of cell motility and invasion.引言 壹、癌細胞轉移(metastasis)機制 1 貳、癌細胞入侵(invasion)機制 2 一、降低細胞間結合力(Disruption of cell-cell adhesion) 2 二、癌細胞移動能力(tumor cell mobility) 3 a.細胞移動的機制 3 b.細胞突觸:lamellipodia, filopodia 的產生 3 三、Matrix metalloproteinase(MMP)與ECM 的降解 4 a. Matrix metalloproteinase(MMP) family 4 b. MMP 的活化與其作用位置 5 c. MMP 影響細胞癌化之機制 6 參、In vitro invasion assay 6 肆、Integrin signaling 與癌細胞入侵能力之關係 7 一、Focal adhesion kinase(FAK)之蛋白結構與focal adhesion assembly 8 二、FAK 對Rho family 的調控 9 三、FAK 對focal adhesion disassembly 的調控 10 伍、癌細胞衍生轉移能力之模式 10 陸、實驗目的 11 材料與方法 壹、Cell culture and antibody 18 貳、Higher invasive potential cell selection, In vitro Invasion assay and haptotaxis migration assay 18 參、Growth experiment 19 肆、Cell lysis and Western blotting 20 伍、Immunoprecipation 22 陸、Gelatin zymography 22 柒、Cell-ECM attachment and spreading assay 23 捌、Wound healing migration assay 24 ii 玖、Immunofluorescence 24 拾、FACSC for cell size test 25 結果 壹、確認篩選出之A431 III 具有更強的入侵能力 26 貳、MMPs 對A431 III 入侵能力之影響 27 參、比較A431 P 與A431 III 之細胞移動能力 28 肆、A431 III 形成穩定之lamellipodia 結構，助其快速移動 29 伍、比較A431 P 與A431 III 之細胞延展性 30 陸、A431 III 癌細胞內FAK 的活性變化 31 討論 43 引用文獻 472232964 bytesapplication/pdfen-US入侵性A431癌細胞之篩選MMP-9 FAK invasionotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52757/1/ntu-94-R92b46038-1.pdf