臺灣博碩士論文加值系統

　　肺癌是全世界發病率和死亡率最高的惡性腫瘤之一，癌細胞轉移是造成病患死亡的首要原因，inoscavin A是從藥用真菌桑黃Inonotus sanghuang中所分離出的新化合物，本研究首先證實inoscavin A能夠抑制A549和LLC肺癌細胞的黏附、侵襲與遷移能力，實驗結果顯示inoscavin A可降低MMP-2和MMP-9的蛋白表現，以及MMP-9與uPA的活性，另外對TIMP-1和PAI-1的蛋白表現有促進作用。在路徑調控上，inoscavin A抑制FAK的磷酸化，並能夠下調MAPK和PI3K/Akt/ mTOR信號路徑，藉由降低蛋白質的磷酸化態ERK、JNK、p38、PI3K、AKT、mTOR、p70S6K和4EBP1的表現，更進一步利用PI3K/AKT抑制劑LY294002、ERK1/2抑制劑PD98059、JNK1/2 抑制劑SP600125、p38抑制劑SB203580和mTOR抑制劑rapamycin，結果顯示對MMP-2和MMP-9的蛋白表現有抑制作用。因此，本實驗表明inoscavin A抑制肺癌細胞的黏附、侵襲與遷移能力，是藉由下調FAK、MAPK和PI3K/AKT/ mTOR訊息傳遞路徑，減少uPA、MMP-2和MMP-9及增加TIMP-1和PAI-1的蛋白表現，顯示inoscavin A是具有潛力開發成抑制肺癌轉移的新藥價值。

Lung cancer is the leading cause of cancer mortality, and metastasis is a primary cause of cancer death. Inoscavin A is a new compound isolated from a medicinal mushroom Inonotus sanghuang. In this study, we first observed that inoscavin A exerted a dose-dependent inhibitory effect on adhesion, migration, and invasion of the highly metastatic A549 and LLC lung cancer cell lines in the absence of cytotoxicity. These effects were associated with a decreased protein expression of MMP-2 and MMP-9 together with an increased expression of TIMP-1 and PAI-1. Inoscavin A also inhibited the enzymatic activity level of uPA and MMP-9. Moreover, we demonstrated that treatment with inoscavin A reduced phosphorylation of FAK. Further studies showed that inoscavin A repressed the activation of MAPK and PI3K/Akt/mTOR signaling pathway, as evidenced by inhibited the phosphorylation of ERK1/2, JNK1/2, p38, PI3K, AKT, mTOR, p70S6K and 4EBP1. In addition, the treatment of A549 cells with inhibitor specific for PI3K /AKT (LY294002), ERK1/2 (PD98059), JNK1/2 (SP600125), p38(SB203580) and mTOR (rapamycin) decreased the expression of MMP-2 and MMP-9. Taken together, these results suggested that inoscavin A inhibits cell adhesion, migration and invasion by reducing uPA, MMP-2 and MMP-9 activation through the suppression of the FAK, mainly mediated by inhibition of MAPK and PI3K/Akt/mTOR signaling pathways. Additionally, induction of TIMP-1 and PAI-1 expression is at least partially involved in the inhibition of MMP-9 and uPA activation by inoscavin A. These findings identify inoscavin A as a promising novel therapeutic agent for lung cancer.

目錄 I
圖目錄 IV
縮寫表 VII
中文摘要 VIII
Abstract IX
第一章 研究緣起 1
第二章 文獻回顧 2
第一節 肺癌 2
第二節 癌細胞轉移 5
第三節 基質金屬蛋白酶 8
第四節 金屬蛋白酶組織抑制劑 13
第五節 Plasminogen activation system 15
第六節 FAK 17
第七節 MAPK家族 18
第八節 PI3K/AKT/mTOR 信號路徑 22
第九節 藥物簡介 25
第三章 研究方法 30
第一節 實驗架構 30
第二節 實驗材料 31
一、 藥品與試劑 31
二、 儀器設備與器材 34
第三節 實驗方法 36
一、 藥物配製 36
二、 細胞培養 36
三、 細胞存活率試驗 (MTT assay) 40
四、 明膠蛋白酶活性電泳分析 (Gelatin Zymography) 42
五、 酪蛋白-纖維蛋白溶解酶活性電泳分析 45
六、 細胞傷口癒合試驗 (Wound-Healing Assay) 47
七、 細胞移行試驗 (Transwell Migration Assay) 48
八、 細胞侵襲試驗 (Transwell Invasion Assay) 49
九、 細胞黏附試驗 (Adhesion Assay) 51
十、 西方墨點法 (Western Blotting) 53
十一、 抑制劑之處理 59
十二、 統計分析 59
第四章 實驗結果 60
第一節 Inoscavin A對A549及LLC細胞存活率之影響 60
第二節 Inoscavin A對A549及LLC細胞分泌MMP活性之影響 64
第三節 Inoscavin A對A549細胞分泌uPA活性之影響 67
第四節 Inoscavin A對A549及LLC細胞移行能力之影響 69
第五節 Inoscavin A對A549及LLC細胞侵襲能力之影響 74
第六節 Inoscavin A對A549黏附能力之影響 77
第七節 Inoscavin A對A549之MMP-2、MMP-9和TIMP-1蛋白表現
量的影響 79
第八節 Inoscavin A對A549之PAI-1蛋白表現量的影響 84
第九節 Inoscavin A對A549之p-FAK蛋白表現量的影響 86
第十節 Inoscavin A對A549細胞之MAPK家族蛋白表現量的影響 88
第十一節 Inoscavin A對A549細胞之PI3K/AKT/mTOR路徑蛋白表現
量的影響 93
第十二節 A549細胞投予PI3K/AKT抑制劑LY294002、ERK1/2抑制
劑PD98059、JNK1/2 抑制劑SP600125、p38抑制劑
SB203580和mTOR抑制劑Rapamycin之MMP-2和MMP-9
之蛋白表現量的影響 101
第五章 討論 105
第六章 結論 111
第七章 參考文獻 113

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