臺灣博碩士論文加值系統

HGF (Hepatocyte growth factor) (肝細胞生長因子)是癌細胞轉移的必要因素之一，能夠促進肝癌細胞腫瘤發展。c-Met，HGF的RTK (Receptor tyrosine kinase) (受體酪胺酸激酶) 被認為是治療肝細胞癌的潛在治療標靶。所以才更迫切需要去探討HGF/ c-Met訊息的詳細機制，從而制定更有效的標靶策略。
我們最近研究證實Hep G2細胞移動所需的HGF誘導ERK- paxillin訊息波動。我們進一步發現HGF誘導c-Met, JNK及paxillin (Ser178)訊息磷酸化，在初始波峰的0.5小時、在中間下降的4小時和之後的高峰10小時是共同的波動模式特徵。
這樣的波動訊息使人聯想到c-Met的內吞作用，以時間及空間的方式控制訊息傳導。HGF誘導RTK內吞作用後，訊息傳導可以在早期內涵體中持續後再回收至漿膜上或是被標上泛素直接送到溶酶體降解。
在本篇中，我們證實了內涵體形成在0.5小時增強JNK/ Paxillin (Ser178)訊息磷酸化所必須。另一方面，以Dynasore 阻斷早期內涵體形成，可能抑制HGF誘導Hep G2細胞移動和JNK/ Paxillin訊息。
我們最近研究亦有證實蛋白質激酶C可調控Hep G2細胞移動中HGF誘導的ERK/ JNK- paxillin訊息波動。所以我們進一步的探討HGF誘導訊息波動是與PKC所調控的c-Met內吞作用有關。隨後，PKC 調控 c-Met內涵體降解之後訊息下降直到4小時。在10小時，JNK/ Paxillin (Ser178) 訊息磷酸化上升，可能是經由Golgi-localized, gamma – ear – containing , Arf -binding proteins 3 (GGA3) 調控內涵體的再回收。
最後，HGF誘導Hep G2細胞移動和轉移可經由內吞作用抑制劑被抑制，與內涵體相關的分子是否有希望可以作為預防HGF誘導肝細胞癌發展的標靶治療。

Hepatocyte growth factor (HGF) is one of the essential metastatic factors capable of triggering tumor progression of hepatocellular carcinoma cell (HCC). c-Met, the receptor tyrosine kinase (RTK) of HGF was regarded as a potential therapeutic target for treatment of hepatocellular carcinoma. It is urgent to explore the detailed mechanisms of HGF/c-Met signaling for devising more effective targeting strategy.
Our recent study demonstrated that HGF-induced fluctuant ERK-paxillin signaling for cell migration of HepG2. We further found HGF-induced phosphorylations of c-Met, JNK and paxillin (Ser178) shared a common fluctuant pattern characterized by an initial peak at 0.5h, a middle drop at 4h and a later peak at 10 h.
Such signaling fluctuation was reminiscent of c-Met endocytosis, which control signal transduction in a temporal and spatial manner. After HGF-induced RTK endocytosis, signal transduction can be sustained within early endosome which may recycle back to the plasma membrane or subject to ubiquitin - directed lysosomal degradation.
Here, we demonstrated that endosome formation is required for initial enhancement of JNK/Paxillin (S178) phosphorylations at 0.5 h. On the other hand, blockade of early endosome formation by dynasor may suppress HGF-induced cell migration of HepG2 and suppress HGF- induced fluctuant JNK-paxillin signaling.
Our recent study demonstrated that protein kinase C (PKC) mediated HGF-induced fluctuant ERK/JNK-paxillin signaling for cell migration of HepG2. We further proposed that HGF-induced fluctuant signaling is well correlated with endocytosis of c-Met, mediated by PKC. Subsequently, PKC mediates endosomal c-Met degradation for subsequent signal declination until 4h. At 10 h, the phosphorylations of JNK/ Paxillin (S178) rised again via Golgi-localized, gamma – ear – containing , Arf -binding proteins 3 (GGA3)-mediated endosomal recycling.
Finally, HGF-induced cell migration and metastasis of HepG2 can be prevented by inhibitors of endocytosis, implicating that critical endosomal components are promising therapeutic targets for preventing HGF-induced tumor progression of HCC.

目錄
Abstract IV
中文摘要 VI
第一章 緒論 1
1.1 研究背景 1
1.2研究目的： 5
第二章 研究方法 6
2.1 人類肝癌細胞的培養 (Cell culture) 6
2.2 西方墨點法 (Western Blot) 6
2.3 細胞移動之測定 (Migration) 11
2.4 小髮夾彎RNA (Small hairpin RNA; shRNA) 12
2.5 轉染細胞 (Transient Transfection) 14
2.6 小干擾RNA（Small interfering RNA；siRNA）轉染細胞 15
2.7 免疫螢光分析 (Immunofluorescence assay) 16
2.8 侵犯試驗 (Invasion assay) 17
2.9 建立肝內轉移動物模式 (Establisment of intrahepatic metastatic in animal model) 18
第三章 結果 19
3.1 HGF誘導c-Met-JNK-Paxillin訊息波動 19
3.2 網格蛋白依賴性 (clatherin dependent) 內涵體 (endosome) 形成是JNK,Paxillin磷酸化初始波動所必須的 20
3.3 PKC在HGF誘導訊息波動初期時負調控JNK/Paxillin(Ser178) 磷酸化 21
3.4 PKC及PKC負責HGF誘導c-Met降解及抑制JNK / Paxillin (Ser178) 訊息 21
3.5 HGF誘導p-JNK/ p-Paxillin (S178) 與GGA3 colocalization是JNK / Paxillin (S178) 所需 22
3.6 HGF在PKC依賴性模式下誘導GGA3磷酸化 23
3.7 HGF誘導內吞作用是HCC腫瘤發展所必需的 24
第四章 討論 27
4.1 網格蛋白-依賴性內涵體形成是在HGF處理早期時JNK/Paxillin完全活化所必須 27
4.2 PKC  及 PKC  調控c- Met降解是與抑制JNK - Paxillin訊息相關 27
4.3 GGA3調控內涵體再回收再度活化JNK-Paxillin(S178)訊息 28
4.4 c-Met內吞作用在HCC發展中的角色 28
第五章 結論 29
第六章 參考文獻 30

圖目錄
圖 1 36
圖 1 A. 在12小時中HGF 誘導Met / JNK/ Paxillin (Ser 178) 訊息波動 36
圖 1 B.圖1A的量化結果 36
圖2. 38
圖 2. PKC和clathrin-dependent 內涵體形成調控JNK/ paxillin (Ser178) 磷酸化的差異 38
圖 3. 39
圖 3 A. PKC  / 是HGF誘導c-Met降解和負調控JNK / paxillin (Ser178) 磷酸化所必須 39
圖 3 B. PKC isozymes 轉染效果 39
圖4. 41
圖 4. HGF誘導p-JNK/p-Paxillin (Ser178)與GGA3 colocalization在10 hours的時候 42
圖5. 43
圖 5A . GGA3 siRNA轉染效果 43
圖 5B . GGA3 是HGF誘導JNK/Paxillin (Ser178) 磷酸化在0.5及10 hours時所必須的 43
圖 6 45
圖 6 A. 在12小時中HGF 誘導GGA3訊息波動 45
圖 6 B. PKC可調控HGF所誘導的GGA3磷酸化 46
圖 6 C . PKC調控HGF誘導GGA3磷酸化 47
圖 7 48
圖 7 A . HGF誘導的內吞作用是HCC移動時所需的 48
圖 7 B . HGF誘導的內吞作用是HCC侵犯時所需的 49
圖 7 C . HGF誘導的內吞作用是HCC 肝內轉移時所需的 50

表目錄
表格 1. Western blot膠的濃度圖表 47
表格 2. 抑制劑圖表 48
表格 3. shRNA sequence 圖表 49
表格 4. 訊息傳導路徑圖表 50

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