臺灣博碩士論文加值系統

胰臟癌為全世界癌症死亡率第四名，為人類最惡性的腫瘤之一。由於晚期診斷及對目前臨床治療藥物反應不良，其五年存活率約為3-5%。蜂膠素 C (propolin C)，為臺灣蜂膠中所分離出的活性物質，過去文獻指出蜂膠素 C 具有抗腫瘤活性，但對於胰臟癌細胞的抗癌機制尚未明瞭。本研究目的主要探討蜂膠素 C 對胰臟癌細胞抗癌作用及其相關機轉的影響。結果顯示，蜂膠素 C 能夠抑制胰臟癌細胞株Kp4及較具有轉移型態的細胞 Kp4 liver-meta 的生長。在細胞群形成試驗中 (Colony formation assay) 中，給予蜂膠素 C 處理下，會隨著劑量增加而抑制細胞群生長。流式細胞儀分析中，Kp4 liver-meta 細胞較 Kp4 細胞顯著停滯於 Sub-G1 期。在Kp4 liver-meta細胞中，發現蜂膠素 C 會抑制表皮生長因子 (epidermal growth factor receptor, EGFR) 表現及其下游路徑 AKT 及 ERK 的活化。同時，EGFR的降解並不是經由轉錄作用的抑制，而是影響其蛋白穩定性。我們也發現蜂膠素C 能誘導自噬作用指標蛋白 LC3 的表現。處理自噬作用抑制劑 (Chloroquine 及 3-Methyladenine) 時，蜂膠素 C 抑制 EGFR 表現有顯著的回升。蜂膠素 C 也會增加 AMP 蛋白激酶 (AMP-activated protein kinase , AMPK) 的活化。使用AMPK 抑制劑 (Compound C) 時，蜂膠素 C 抑制 EGFR 表現也會回升。由以上結果顯示，蜂膠素C 在高度轉移的胰臟癌細胞中，藉由活化 AMPK 相關訊息路徑，進而誘導細胞自噬作用使 EGFR 蛋白降解而導致細胞凋亡，達到抑制胰臟癌細胞生長效果。

Pancreatic cancer is the fourth leading cause of cancer-related deaths worldwide and is one of the most aggressive and devastating human malignancies. The overall 5-year survival rate is 3~5% cause of advanced stage at diagnosis and poor response to current treatment. Searching the active compound to prevent or treat pancreatic cancer is urgency. Propolin C, a bioactive compound from Taiwanese propolis, has been demonstrated to possess anti-tumor activity. However the anti-cancer mechanism of propolin C on pancreatic cancer is still unclear. In the present study, the anti-cancer molecular mechanism of prpolin C were evaluated in pancreatic cancer. The results showed that cell viability was inhibited by propolin C in a dose- and time-dependent manner in Kp4 and Kp4 liver-meta cells. The number of surviving colonies was significantly reduced by propolin C. Apoptotic cells were significantly accumulated by propolin C treatment in Kp4 liver-meta cells than parental cells. Propolin C inhibited the expression of epidermal growth factor receptor (EGFR) and EGFR-mediated AKT and ERK activation. Interestingly, down-regulation of EGFR expression was trough disruption protein stability rather than transcriptional regulation and only observed in Kp4 liver-meta cells. Furthermore, propolin C induced autophagy molecular, LC3, expression. Propolin C- downregulated EGFR expression was reversed by autophagy inhibitors, chloroquine (CQ) and 3-Methyladenine (3MA), treatment. Meanwhile, propolin C also induced AMP-activated protein kinase (AMPK) activation. And inhibiting AMPK activation by pharmacological inhibitor, compound C, reversed propolin C-downregulated EGFR expression. Accordingly, the present results revealed the propolin C induced highly metastasis pancreatic cell apoptosis might be through AMPK-mediated autophagy following by EGFR degradation.

中文摘要 ………………………………………………………………………….. i
Abstract …………………………………………………………………………… ii
致謝 ………………………………………………………………………………. iii
縮寫表 ……………………………………………………………………………. iv
第一章、文獻回顧 ……………………………………………………………….. 1
一、胰臟癌 (pancreatic cancer) ……………………………………………..….. 1
(一)、概論 ……………………………………………………………………...… 1
(二)、胰臟癌的分類與病理 ……………………………………………………... 1
(三)、胰臟癌移行與侵襲能力 ……………………………………………..……. 1
(四)、臨床上常用的治療方法 ……………………………………………..……. 2
二、表表皮生長因子受體 (Epidermal growth factor receptor, EGFR) ……... 3
(一)、表皮生長因子受體活化與下游路徑 ………………………………..……. 4
(二)、表皮生長因子與自噬作用 ……………………………………………..…. 4
三、自噬作用 (Autophagy) ……………………………………………..………. 5
(一)、自噬作用機制 ……………………………………………..…..………..…. 5
(二)、 AMP蛋白激酶 (AMP activated protein kinase , AMPK) ………………. 6
(三)、哺乳動物雷帕黴素靶蛋白 (mTOR) ……………………………………… 7
四、臺灣蜂膠 (Taiwanese propolis) …………………………………………… 8
(一)、蜂膠的來源與分類 ………………………………………..……………… 8
(二)、蜂膠之生理功能 ………………………………………….….…………… 9
(三)、臺灣蜂膠與癌症之間的關係 ………………………………..…………… 9
1. 誘導細胞凋亡、自噬作用 …………………………………………....……… 9
2. 抗腫瘤生長 …………………………………………………………………… 10
3. 抗移行及侵襲能力 …………………………………………………………… 10
第二章、研究設計與目的 …………………………………………………….…. 11
(一)、研究目的 ………………………………………………………………….. 11
(二)、實驗設計 ………………………………………………………………… 11
第三章、材料與方法 ………………………………………………………..…… 14
一、臺灣蜂膠萃取物蜂膠素C ……………………………………………..…… 14
二、實驗材料 ………………………………………………………………..…… 14
(一) 細胞株 ………………………………………………………………..…....… 14
(二) 培養基 ………………………………………………………………..……… 14
(三) 藥品試劑 ……………………………………………………………..……… 15
(四) 抗體 …………………………………………………………………..…..…. 17
三、實驗儀器 ……………………………………………………………….….... 18
四、實驗耗材 ………………………………………………………………...….. 19
五、常用容液配置 ………………………………………………………..…….. 20
六、實驗方法………………………………………………………………….…. 24
七、統計分析 …………………………………………………………………… 31
第四章、實驗結果 ……………………………………………………………..… 32
(一)、胰臟癌細胞株Kp4及Kp4 liver-meta 移行能力及EMT標記蛋白表現 .. 32
(二)、胰臟癌細胞株Kp4及Kp4 liver-meta之EGFR相關訊息傳遞路徑及自噬作用標的蛋白表現 …………………………………………………………………. 32
(三)、臨床藥物Gemcitabine對Kp4及Kp4 liver-meta 細胞存活率之影響….. 33
(四)、蜂膠素C對Kp4及Kp4 liver-meta 細胞存活率影響 ……………..…… 33
(五)、蜂膠素C對Kp4及Kp4 liver-meta集落形成之影響 ………………...… 33
(六)、蜂膠素C對Kp4及Kp4 liver-meta 細胞週期影響 …………………..… 34
(七)、蜂膠素C對胰臟癌細胞Kp4 liver-meta細胞凋亡、自噬作用的蛋白表現 ………………………………………………………………………..………… 35
(八)、蜂膠素C對胰臟癌細胞Kp4及Kp4 liver-meta之EGFR相關路徑及自噬作用標的蛋白影響之比較 ………………………………………………..……… 35
(九)、蜂膠素C對胰臟癌細胞Kp4及Kp4 liver-meta之EGFR mRNA影響之比較 ……………………………………………………………………………..…… 36
(十)、蜂膠素C藉由自噬作用路徑降低Kp4 liver-meta的EGFR表現 ……… 36
(十一)、自噬作用抑制劑對蜂膠素C影響Kp4 liver-meta細胞存活率之效果 ……………………………………………………………………..…………… 37
(十二)、蜂膠素C對Kp4 liver-meta細胞週期之自噬作用抑制劑介入下的影響 ………………………………………………………………………..……….… 37
(十三)、蜂膠素C對胰臟癌細胞Kp4 liver-meta之p-EGFR、p-AKT、p-ERK、p-AMPK及p-mTOR蛋白表現影響 …………………………………………….… 38
(十四)、蜂膠素C藉由p-AMPK路徑降低EGFR表現………………………… 38
(十五)、蜂膠素C對Kp4 liver-meta細胞存活率之AMPK抑制劑介入下的抑制效果 ……………………………………………………………………………… 39
(十六)、蜂膠素C藉由Ubiquitin /proteasome路徑降低EGFR表現 …..….… 39
第五章、討論 …………………………………………………………………… 41
(一)、胰臟癌細胞轉移與化療抗藥性相關機制之探討 ……………….……… 41
(二)、蜂膠素C抑制胰臟癌細胞生長之訊息傳遞路徑之探討 …………..…… 43
1.EGFR下游訊息傳遞路徑 ……………………………………………..…… 44
2.自噬作用與AMPK訊息傳遞路徑 ……………………………….………… 46
(三)、總結 ……………………………………………………………..………… 47
第六章、圖表 …………………………………………………………………… 48
第七章、參考文獻 ……………………………………………………………… 69
第八章、附圖 …………………………………………………………………… 75
第九章、附表 …………………………………………………………………… 80
圖目錄
圖一 、胰臟癌細胞株Kp4及Kp4 liver-meta 移行能力及EMT標記蛋白表現 ………………………………………………………………………………… 48
圖一 (C)、胰臟癌細胞株Kp4及Kp4 liver-meta之EGFR相關訊息傳遞路徑及自噬作用標的蛋白表現 …………………………………………………...………50
圖二、臨床藥物Gemcitabine對Kp4及Kp4 liver-meta 細胞存活率之影響. …51
圖三、蜂膠素C對Kp4及Kp4 liver-meta 細胞存活率影響 …………….…… 52
圖四、蜂膠素C對Kp4及Kp4 liver-meta集落形成之影響 ………..………… 54
圖五、蜂膠素C對Kp4及Kp4 liver-meta 細胞週期影響 …………….……… 55
圖六、蜂膠素C對胰臟癌細胞Kp4 liver-meta細胞凋亡及自噬的蛋白表現 ... 56
圖七、蜂膠素C對胰臟癌細胞Kp4及Kp4 liver-meta之EGFR相關路徑及自噬作用標的蛋白影響之比較 ………………………………………………………. 57
圖八、蜂膠素C對胰臟癌細胞Kp4及Kp4 liver-meta之EGFR mRNA影響之比較 ……………………………………………………………………………….… 58
圖九、蜂膠素C藉由自噬作用路徑降低Kp4 liver-meta的EGFR表現 …..… 59
圖十、自噬作用抑制劑對蜂膠素C影響Kp4 liver-meta細胞存活率之效果 ………………………………………………………………………….……… 60
圖十一、自噬作用抑制劑對蜂膠素C影響細胞週期之效果……..……………. 61
圖十二、蜂膠素C對胰臟癌細胞Kp4 liver-meta之p-EGFR、p-AKT、p-ERK、p-AMPK及p-mTOR蛋白表現影響 ……………………………………….…… 62
圖十三A、蜂膠素C藉由p-AMPK路徑降低EGFR表現 …………………… 63
圖十三B、Metformin藉由p-AMPK路徑降低EGFR表現 ……………...……64
圖十四、蜂膠素C對Kp4 liver-meta細胞存活率之AMPK抑制劑介入下的抑制效果 ……………………………………………………………………….……… 65
圖十五、蜂膠素C藉由Ubiquitin /proteasome路徑降低EGFR表現 …..…… 67
圖十六、蜂膠素C抑制胰臟癌細胞生長之機制圖 …………………………… 68
 
附圖目錄
附圖一、上皮間質轉換 ………………………………………………………… 75
附圖二、EGFR調節細胞存活的訊息傳遞路徑 ……………………………… 76
附圖三、自噬作用的過程 ……………………………………………………… 77
附圖四、AMP蛋白激酶訊息傳遞路徑 ………………………………………… 78
附圖五、mTOR訊息傳遞路徑 …………………………………………….…… 79
附表目錄
附表一、不同種類之蜂膠具有之活性的化合物 ………………………….…. 80
附表二、不同產的之臺灣蜂膠中蜂膠素C、D、F 含量 …………………… 81

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