臺灣博碩士論文加值系統

斑蝥素是莞菁 (斑蝥，一種傳統的中藥) 的組成成分之一，為?衛?化合物的一種。目前已被證實，斑蝥素具有抗腫瘤、抗菌、防病毒和促進免疫活動的能力。無數研究已報告指出斑蝥素能誘導許多人類癌症細胞的細胞膜出泡、caspase 活化、DNA 斷裂，以及細胞週期的停滯。然而，沒有研究指出，斑蝥素能夠誘導人類上皮癌 A431 細胞凋亡。在本研究中，我們將調查 A431 細胞的細胞週期停滯和凋亡的分子機制。我們發現斑蝥素能夠使 A431 細胞的存活率下降，並有活性氧化物質 （ROS） 和胞內鈣離子 （Ca2 +） 增加的現象，粒腺體膜電位 （ΔΨm） 的降低及誘導 A431 細胞產生凋亡的現象。 斑蝥素也增加細胞凋亡途徑的蛋白表現量，如： Caspase-3、Caspase-8、Caspase-9、PARP 的活化、cytochrome c 和 Bax，但是 Mcl-1 和 Bcl-2 蛋白表現量的現象則有下降的趨勢。Annexin V的親和力檢測則顯示，A431 細胞對於凋亡顯著的增加有時間的依存性，並且經由 JNK 介導的 c-Jun 訊號路徑調控蛋白的表現量。我們使用特殊的 caspase 抑制劑預處理 A431 細胞，經處理斑蝥素後結果顯示，其存活率較單獨處理斑蝥素時有回升的現象。根據這些觀察，我們推測斑蝥素能經由活化 caspase 的依賴訊號路徑，誘導 A431 細胞產生細胞凋亡的現象。

Cantharidin (CTD) is one of the component from a traditional Chinese medicine named mylabris, and it is a type of terpenoid. It has been demonstrated that CTD possesses antitumor, antibiotic, antivirus and promotes immunity activities. Many studies have reported that CTD induced membrane blebbing, caspase activation, DNA fragmentation, cell cycle arrest in many human cancer cell lines. However, there is no report showing CTD induced apoptosis in human epithelial carcinoma A431 cells. In this study, we investigated the molecular mechanisms of apoptosis in A431 cells after exposed to CTD. We found that CTD decreased the percentage of cell viability, increased the levels of reactive oxygen species (ROS) and calcium (Ca2+) productions, decreased the level of mitochondrial membrane potential (ΔΨm) and triggered apoptosis in A431 cells. CTD increased the protein levels of cell death pathway such as the Caspase-3, -8, -9, PARP active form, cytochrome c and Bax, decreased the anti-apoptosis protein levels such as Mcl-1 and Bcl-2. Annexin V affinity assay was performed by flow cytometry and our results indicated that CTD significantly induced apoptosis in A431 cells in a time-dependent manner and it promoted the protein levels of JNK-mediated c-Jun signaling pathway. We used the specific caspase inhibitors to pre-treat A431 cells, and then exposed to CTD and results showed that CTD increased percentage of viable cells when compared to CTD treatment alone in A431 cells. Based on those observations, we suggest that CTD can induce apoptosis in A431 cells through the activation of caspase-dependent signaling pathways.

總目錄
總目錄---------------------------------------------------------------------I
圖目錄-------------------------------------------------------------------VII
表目錄---------------------------------------------------------------------X
致謝----------------------------------------------------------------------XI
縮寫表--------------------------------------------------------------------XII
中文摘要------------------------------------------------------------------XIV
英文摘要-------------------------------------------------------------------XV
第一章、前言-----------------------------------------------------------------1
第一節、研究背景----------------------------------------------------------1
一、癌症的生成--------------------------------------------------------1
二、人類上皮鱗狀癌 A431 細胞之介紹---------------------------------------2
三、斑蝥素 (Cantharidin) 之介紹----------------------------------------3
四、細胞凋亡 (Apoptosis) 之介紹----------------------------------------5
(一)、內在路徑 (Intrinsic pathway)--------------------------------5
(二)、外在路徑 (Extrinsic pathway)--------------------------------6
(三)、內質網壓力 (Endoplasmic Reticulum Stress)--------------------8
五、Mitogen-activated protein kinases (MAPKs) 之介紹------------------9
第二節、研究目的--------------------------------------------------------------10
第二章、研究方法--------------------------------------------------------------11
第一節、研究材料--------------------------------------------------------------11
一、細胞株來源--------------------------------------------------------11
二、藥品試劑----------------------------------------------------------11
三、儀器設備與器材-----------------------------------------------------16
第二節、研究設計---------------------------------------------------------------17
一、實驗設計流程---------------------------------------------------------------17
二、實驗方法-------------------------------------------------------------------18
(一)、藥物的配製 (斑蝥素；Cantharidin)--------------------------------------18
(二)、人類上皮鱗狀癌細胞株 (A431 cell) 細胞培養-------------------------------18
(三)、細胞的冷凍保存--------------------------------------------------------19
(四)、冷凍細胞的活化--------------------------------------------------------20
(五)、細胞存活率 (Cell Viability)------------------------------------------21
(六)、Annexin V affinity assay-------------------------------------------22
(七)、彗星試驗 (Comet assay)----------------------------------------------23
(八)、細胞核質濃縮現象 (DAPI 染色)-------------------------------------------26
(九)、DNA膠體電泳 (Agarose gel electrophoresis)---------------------------27
(十)、細胞週期 (Cell cycle) 分析-------------------------------------------30
(十一)、鈣離子 (Ca2+) 釋出的變化--------------------------------------------32
(十二)、活性氧化物質 (ROS) 產生之檢測----------------------------------------33
(十三)、粒線體膜電位 (ΔΨm) 之檢測-------------------------------------------34
(十四)、Caspase-3 活性測試 (Caspase-3 activity)---------------------------35
(十五)、Caspase-8 活性測試 (Caspase-8 activity)---------------------------37
(十七)、西方墨點法 (Western blot)------------------------------------------38
(十八)、活性氧化物質清除劑 (ROS scavenger) 之檢測----------------------------42
(十九)、鈣離子螯合劑 (Ca2+ chelator) 之檢測---------------------------------43
(二十)、粒線體膜電位抑制劑之檢測---------------------------------------------44
(二十一)、Caspase 抑制劑之檢測---------------------------------------------45
(二十二)、免疫螢光染色 (Immunofluorescence staining)-----------------------46
(二十三)、即時定量 PCR (Quantitative PCR Analysis)------------------------47
(二十四)、活體試驗--------------------------------------------------------49
(二十五)、統計分析--------------------------------------------------------50
第三章、研究結果--------------------------------------------------------------51
第一節、斑蝥素對人類上皮鱗狀癌 A431 細胞型態之變化----------------------------------51
第二節、斑蝥素對人類上皮鱗狀癌 A431 細胞存活率之影響--------------------------------52
第三節、斑蝥素對人類上皮鱗狀癌 A431 細胞誘導凋亡之影響------------------------------53
第四節、觀察斑蝥素對人類上皮鱗狀癌 A431 細胞之 DNA 受損影響-------------------------55
一、彗星試驗 (Comet assay)---------------------------------------------------55
二、DAPI 染色法 (DAPI staining)----------------------------------------------56
三、DNA膠體電泳 (DNA gel electrophoresis)-------------------------------------58
第五節、觀察斑蝥素對人類上皮鱗狀癌 A431 細胞之細胞週期影響---------------------------60
第六節、以西方墨點法探討斑蝥素對人類上皮鱗狀癌 A431 細胞週期蛋白之表現-----------------62
第七節、斑蝥素對人類上皮鱗狀癌 A431 細胞之活性氧化物質 (Reactive oxygen species, ROS) 之影響------------------------63
第八節、斑蝥素對人類上皮鱗狀癌 A431 細胞之粒線體電位 (Mitochondrial membrane potential, ΔΨm) 的影響-----------------65
第九節、斑蝥素對人類上皮鱗狀癌 A431 細胞活化 Caspase-3 的影響-----------------------------------------------------67
第十節、斑蝥素對人類上皮鱗狀癌 A431 細胞活化 Caspase-9 的影響----------------------------------------------------69
第十一節、斑蝥素對人類上皮鱗狀癌 A431 細胞活化 Caspase-8 的影響--------------------------------------------------71
第十二節、以西方墨點法探討斑蝥素對人類上皮鱗狀癌 A431 細胞凋亡蛋白與細胞週期相關蛋白的表現變化---------------------------73
第十三節、斑蝥素對預處理 ROS 清除劑之人類上皮鱗狀癌 A431 細胞存活率的影響--------------------------------------------76
第十四節、斑蝥素對預處理粒線體膜電位抑制劑之人類上皮鱗狀癌 A431 細胞存活率的影響-----------------------------------------77
第十五節、斑蝥素對預處理 caspase 抑制劑之人類上皮鱗狀癌 A431 細胞存活率的影響-----------------------------------------78
第十六節、斑蝥素對人類上皮鱗狀癌 A431 細胞內鈣離子 (Ca2+) 釋出的影響-------------------------------------------------80
第十七節、以西方墨點法探討斑蝥素對人類上皮鱗狀癌 A431 細胞內質網壓力路徑之相關調控蛋白表現量變化----------------------------82
第十八節、斑蝥素對預處理鈣離子螯合劑之人類上皮鱗狀癌 A431 細胞存活率的影響----------------------------------------------84
第十九節、以免疫螢光染色探討斑蝥素對人類上皮鱗狀癌 A431細胞中凋亡相關蛋白表現與轉移之影響----------------------------------85
第二十節、以西方墨點法探討斑蝥素對人類上皮鱗狀癌 A431 細胞內 MAPK 路徑之相關調控蛋白表現量變化----------------------------87
第二十一節、以 Quantitative PCR Analysis 探討斑蝥素對人類上皮鱗狀癌 A431 細胞之凋亡相關蛋白其mRNA表現------------------89
第二十二節、利用活體實驗檢測斑蝥素對人類上皮鱗狀癌 A431 細胞異位移植腫瘤抑制之能力---------------------------------------90
第四章、討論--------------------------------------------------------------------------95
第五章、結論-------------------------------------------------------------------------100
第六章、參考文獻----------------------------------------------------------------------102

圖目錄
圖一：民國 99 及 100 年台灣主要十大死因-------------------------------1
圖二：民國 99 及 100 年台灣主要十大癌症死亡率--------------------------2
圖三：斑蝥素 (Cantharidin) 之結構式---------------------------------3
圖四：Apoptosis 與 Necrosis 之不同---------------------------------7
圖五：外在、內在與其他相關凋亡路徑圖---------------------------------- 7
圖六：哺乳動物細胞中 UPR 訊號傳導路徑相關圖----------------------------9
圖七、本實驗研究流程圖----------------------------------------------17
圖八、利用倒立式相位差顯微鏡觀察 A431 細胞經斑蝥素處理 48 小時後細胞型態之變化-------------------------51
圖九、以 MTT assay 方法檢測當以斑蝥素處理 A431 細胞經過 24及 48 小時後細胞之存活率-------------------52
圖十、A431 細胞株經斑蝥素處理後細胞凋亡現象-------------------------------------------------------54
圖十一、不同濃度之斑蝥素對於 A431 細胞經處理後其 DNA 的片段拖尾程度----------------------------------56
圖十二、不同濃度之斑蝥素對於 A431 細胞處理後，其細胞內染色質凝集之現象---------------------------------58
圖十三、以不同濃度之斑蝥素處理 A431 細胞株後 DNA 斷裂之影響-----------------------------------------59
圖十四、A431 細胞株經斑蝥素處理後細胞週期的之影響變化圖與量化圖--------------------------------------61
圖十五、A431 細胞株經斑蝥素處理後，細胞週期相關調控蛋白之蛋白表現量變化--------------------------------62
圖十六、以流式細胞儀觀察 A431 細胞株經斑蝥素處理後 ROS 在細胞內的表現---------------------------------64
圖十七、以流式細胞儀觀察 A431 細胞株經斑蝥素處理後粒線體膜電位所受之影響-------------------------------66
圖十八、斑蝥素造成 A431 細胞活化 Caspase-3 的影響-------------------68
圖十九、斑蝥素造成 A431 細胞活化 Caspase-9 的影響-------------------70
圖二十、斑蝥素造成 A431 細胞活化 Caspase-8 的影響-------------------72
圖二十一、當 A431 細胞株經斑蝥素處理後以西方墨點法觀察粒線體路徑之相關蛋白表現量之變化---------------------75
圖二十二、斑蝥素對預處理 N-acetyl cysteine 之 A431 細胞的影響-------------------------------------76
圖二十三、斑蝥素對預處理 CsA 之 A431 細胞的影響----------------------- ----------------------------77
圖二十四、斑蝥素對預處理 Caspase 抑制劑之 A431 細胞存活率的影響--------------------------------------79
圖二十五、以流式細胞儀觀察A431 細胞株經斑蝥素處理隨時間的增加 Ca2+ 濃度的影響---------------------------81
圖二十六、斑蝥素對 A431 細胞內質網壓力路徑之相關調控蛋白表現量變化-------------------------------------83
圖二十七、斑蝥素對預處理 BAPTA-AM 之 A431 細胞的影響------------- ---------------------------------84
圖二十八、以免疫螢光染色探討斑蝥素對 A431細胞中凋亡相關蛋白表現與轉移之影響-------------------------------86
圖二十九、斑蝥素對 A431 細胞內 MAPK 路徑之相關調控蛋白表現量變化--------------------------------------88
圖三十、以Quantitative PCR Analysis 探討斑蝥素對 A431 細胞之凋亡相關蛋白其mRNA表現-------------------89
圖三十一、檢測斑蝥素對 A431 細胞異位移植腫瘤抑制能力之活體試驗-----------------------------------------94
圖三十二、斑蝥素誘導人類上皮鱗狀癌 A431 細胞株細胞凋亡可能路徑圖----------------------------------------101

表目錄
表一、Comet assay 使用之 buffer 配置----------------------------------------------25
表二、Agarose Gel 之配置----------------------------------------------------------29
表三、Cell cycle 使用所需之 PI 染劑配置----------------------------------------------31
表四、SDS-PAGE 下層膠 (Separating gel) 之配置與組成---------------------------------40
表五、SDS-PAGE 上層膠 (Stacking gel) 之配置與組成-----------------------------------41
表六：電泳緩衝液 (Electrophoresis running buffer)---------------------------------41
表七：轉漬緩衝液 (Transfer buffer)-----------------------------------------------41

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