活性氧(Reactive oxygen species；ROS)複雜的參與在腫瘤的轉移過程中，特別是在腫瘤細胞的移動、侵犯的階段。已知有一些生長因子(TGF、HGF)、Integrin engagement會誘導ROS的生成啟動腫瘤細胞的轉移。
在過去我們實驗室的研究中指出腫瘤促進劑TPA會促進ROS的生成，ROS與PKC共同作用引發ERK的活化，而ERK的活化則造成肝癌細胞HepG2移動的重要關鍵，由此可見ROS和HepG2移動的相關性。所以本論文主要著重於TPA誘發ROS之生成是否透過integrin所的訊號傳遞，以及TPA所造成之ERK活化是否須ROS及integrin訊號傳遞。首先在不同時間點的實驗中發現TPA誘導HepG2細胞比在懸浮HepG2細胞ROS生成量高，尤其是在1小時有較高量的表現。另外我們也發現穩定表達的PKC的HepG2細胞來觀察其生成的強度都比HepG2細胞高。接著也發現PKC以及integrin訊息傳訊相關的抑制劑都可以減少TPA誘導肝癌細胞株HepG2的ROS生成。根據前面的實驗我們比較貼附細胞與懸浮細胞經過TPA處理後誘導ERK磷酸化的表現，發現貼附細胞ERK磷酸化具有持續的表現但懸浮的細胞沒有這樣的表現，另外我們發現TPA的確會使HepG2細胞產生ROS而促進ERK磷酸化的表現。綜合以上的結果，TPA可能誘導ROS生成是透過 integrin 訊息傳遞途徑以及TPA所造成之ERK活化是須ROS及integrin訊號的傳遞。

Reactive oxygen species (ROS) were involved in tumor metastasis especially at the migration and invasion stage. ROS generation may be induced by growth factors (such as TGFβ and HGF), or integrin engagement, which are capable of triggering tumor progression.
Previous study in our lab indicated that ROS may be generated during migration of hepatoma cell HepG2 induced by tumor promoter TPA in a protein kinase C(PKC) dependent manner. In this study, we further investigated whether integrin-related signal transduction is involved in TPA-induced ROS generation. The flow cytometry was used to analyze ROS generation using DCF-DA as a ROS probe. In the time course study, ROS were found to be generated in HepG2 after treatment of TPA for 0.5 to 6 hr with maximal induction at 1 h. The TPA-induced ROS generation was not observed in cell suspension of HepG2 indicating that integrin triggered signal was requied. Furthermore, we found the PKC inhibitor Bis (bisindolylmaleimides), and inhibitor for the integrin-related signal cascade such as RGD peptide, Src kinase inhibitor, Rac inhibitor (NSC23766) may prevent TPA-induced ROS generation at 1 h. On the other hand, we also found that ROS generation was also required for TPA-induced (ERK) MAPK phosphorylation in attached HepG2 but not HepG2 in cell suspension. Taken together, we suggest that TPA may induce ROS generation via the integrin pathway, which is required for TPA-induced sustained ERK activation and cell migration of HepG2. More detailed mechanisms are still needed to be explored.

目 錄
壹、ABSTRACT 4
貳、中文摘要 5
參、研究背景 6
一.腫瘤的轉移 7
二. 活性氧在腫瘤的轉移中扮演之角色 8
【1】何謂活性氧 (Reactive oxygen species；ROS) 8
【2】活性氧的產生 8
【4】活性氧與訊息傳導的關係 9
【5】活性氧與癌症的關係 10
三. integrin 在腫瘤的轉移中扮演之角色 10
四. PKC(protein kinase C)在腫瘤的轉移中扮演之角色 11
五. TPA促進肝癌細胞HepG2移動的機制 11
肆、研究動機與目的 12
一.肝癌細胞的培養 (Cell culture) 14
二. 西方點墨法 (Western blotting assay) 14
三. 活性氧之測定(Reactive Oxygen Species assay) 18
四.細胞附著試驗(cell adhesion assay) 19
五. Small hairpin RNA(sh RNA) 20
六. Transient transfection 20
陸、研究結果 21
一. TPA激發 HepG2細胞 ROS之生成須透過PKC 22
二. integrin 的訊號傳遞為 TPA在HepG2 細胞中造成ROS的生成所必需 23
三. 啟動integrin signal造成ROS generation 24
四. integrin��ROS訊號途徑與ERK活化之關係 25
柒、討論 26
捌、圖表目錄 28
表格1、10％ SDS-PAGE 膠液配方 29
表格2. 所使用的paxillin sh RNA 種類 29
表格3. transient transfection 試劑與細胞建議使用量 29
圖1. transfection 步驟流程圖 30
圖2. ROS可經由integrin訊息路徑來生成模式 31
圖3. HepG2 細胞經過TPA不同時間處理後ROS生成的表現 33
圖4. HepG2 及PKC stable clone經過TPA處理後ROS生成的表現 34
圖5. PKC抑制劑及integrin-related signal molecules 抑制TPA誘使ROS的生成 37
圖6. paxillin134/135 shRNA 可抑制TPA誘使ROS的生成 38
圖7. 細胞附著後啟動integrin signal誘使ROS的生成 40
圖8. TPA誘使懸浮與貼附的HepG2細胞造成ERK磷酸化之差異 41
圖9. H2O2 generator促進HepG2細胞經過TPA處理後誘導ERK磷酸化 42
圖10. MEK1抑制劑可抑制TPA誘使ROS的生成 43
圖11. TPA與integrin對HepG2細胞作用的訊息傳遞機制（簡圖）。 44
補充圖表 45
S1. paxillin shRNA 可抑制TPA誘使ROS的生成 46
玖、參考文獻 47

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