論文名稱:

Cyclooxygenase-2/Prostaglandin E2路徑參與檳榔子水萃取物處

 

理口腔鱗狀細胞癌細胞株後所誘發的EMT相關表型

 

Involvement of Cyclooxygenase-2/Prostaglandin E2 Pathway

 

in EMT-related Phenotypes in Areca Nut Extract-treated

 

Oral Squamous Cell Carcinoma Cells

研究生:

鄭佳珍  Chia-Chen Cheng

指導教授:

林姝君  Shu-Chun Lin

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

39417008

          學年度:

95

          語文別:

中文

          出版年:

96

關鍵字:

檳榔子水萃取物  ANE

 

上皮細胞轉型成間葉細胞之過程  epithelial-mesenchymal transition

 

環氧化酵素  Cyclooxgensase,COX

 

非類固醇消炎止痛藥  non-steroidal anti-inflammatory drugs,NSAIDs

 

口腔鱗狀上皮癌  Oral squamous cell carcinoma

 

  PGE2

全文說明:

(本論文 20100726 對校內公開)

        論文頁數:

69

摘要:

台灣地區嚼食檳榔人口不斷攀升,口腔癌的發生率及死亡率也逐年增加,過去的研究證實,嚼食檳榔為罹患口腔癌的主因,但檳榔嚼塊的致癌機轉至今仍未釐清。而檳榔嚼塊含多種成份,其中以檳榔子為主要致癌物質,本研究即以檳榔子水萃取物(ANE)為研究對象,來探討口腔癌化的機轉。過去的研究發現,對OECM-1OC3株口腔鱗狀上皮細胞癌細胞處理ANE後,會使cyclooxygenase-2COX-2)的表現增加。COX-2在一般組織是偵測不到的,卻經常大量表現於腫瘤、口腔中癌前及惡性病變中,此外,COX-2可將花生四烯酸轉變成prostaglandin E2PGE2)。而實驗也證明,在正常人類口腔角質細胞中,ANE會增加PGE2的產量。並且,在OECM-1OC3細胞中,ANE尚可誘發epithelial-mesenchymal transitionEMT)相關的型態,包括了:增加vimentinfibronectin的表現以及細胞拉長似纖維母細胞的型態。故vimentinfibronectin的增加也被視為EMT的分子指標。本研究即探討ANE經由COX-2/PGE2路徑誘發EMT相關型態的過程中所扮演的角色及機轉。實驗結果發現在OECM-1OC3種細胞中,ANE會誘發PGE2的產生,且PGE2可進一步誘發vimentin的表現,而在處理COX-2抑制劑NS-398後,由ANE所誘發的vimentin表現會受到抑制,但PGE2卻無法誘發fibronectin的表現。另外,實驗結果也證實,在OC3細胞中,ANE會誘發EP4表現,在OECM-1細胞卻不行。綜合實驗結果,在經過ANE處理的OECM-1OC3細胞中,ANE藉由COX-2/PGE2路徑參與誘發EMT相關型態。而在OC3細胞中,ANE所產生的PGE2很可能是藉由EP4接受器進而誘發EMT相關的型態,但OECM-1則否。故本研究提供初步證據,顯示檳榔透過調升COX-2誘發口腔上皮細胞類似EMT的變化,此對上皮癌化或進行的機轉有參考價值。

 

The areca quid consumption population is increasing in Taiwan, and the incidence and mortality from oral cancer is also ascending. Areca quid chewing has been associated with the high incidence of oral cancer formation. But how areca quid chewing leads to oral cancer remains poorly understand. There are a lot of components in areca quid, and areca nut is one of the major carcinogenic substances of areca quid. The aim of this study was to investigate the possible role of areca nut extract (ANE) in oral carcinogenesis. Previous studies have shown that COX-2 can be up-regulated by ANE in OECM-1 and OC3. COX-2, although not detected in most normal tissues, is often robustly expressed in neoplastic tissues, oral premalignant and malignant lesions. COX-2 can convert arachidonic acid into prostaglandin E2 (PGE2). Studies have shown that prostaglandin production was increased by ANE treatment of normal human oral keratinocytes. Besides, experiments also demonstrated that ANE can induce epithelial-mesenchymal transition (EMT) -related phenotypes including the up-regulation of vimentin and fibronectin and the emergence of fibroblastoid cellular phenotypes in OECM-1 and OC3. Interestingly, vimentin and fibronectin are commonly used as the molecular markers of EMT.

 

In this study, the role of ANE for the induction of EMT-related phenotypes via COX-2/PGE2 pathway was investigated. Pretreatment with COX-2 inhibitor, NS-398, can abrogate the up-regulation of vimentin induced by ANE in both OECM-1 and OC3. Indeed, ANE induced PGE2 production in both cells, and PGE2 consequently induced the expression of vimentin in both cells. However, the expression of fibronectin cannot be induced by PGE2. ANE induced EP4 expression in OC3, but not in OECM-1. These results suggest that COX-2/PGE2 pathway was involved in EMT-related phenotypes in areca nut extract-treated OECM-1 and OC3. Furthermore, PGE2 produced by ANE treatment may induce EMT-related phenotypes through EP4 in OC3, but not in OECM-1. These findings concluded that ANE can induce EMT-related phenotypes by activation of COX-2 signaling, which could be contributive to oral cancer progression.

   

        論文目次:

    頁次

 

中文摘要.......................    1

 

英文摘要.......................    3

 

英文縮寫對照表.........................    5

 

壹、緒論.......................    6

 

一、口腔癌.....................    6

 

二、檳榔嚼塊成分..................    7

 

三、檳榔之致癌性...................    7

 

四、上皮細胞轉型成間葉細胞之過程...........    8

 

五、Vimentin....................    10

 

六、Fibronectin...................    10

 

七、CyclooxygenaseCOX)..............    11

 

八、Cyclooxygenase-2與癌症的關係...........    12

 

九、PGE2與癌症之間的關係...............    14

 

十、PGE2EP4....................    15

 

貳、研究動機.....................    17

 

參、研究目標.....................    17

 

肆、實驗材料.....................    18

 

一、藥品試劑.....................    18

 

二、細胞株......................    19

 

三、藥物配製.....................    20

 

伍、實驗方法.....................    21

 

一、細胞培養.....................    21

 

二、西方墨點法(Western blot)............    22

 

三、免疫螢光染色...................    24

 

四、PGE2含量測定...................    25

 

五、統計.......................    27

 

陸、結果.......................    28

 

一、在OECM-1細胞中ANE可藉由COX-2誘發EMT相關表型..    28

 

二、在OECM-1細胞中ANE可誘發PGE2的分泌........    28

 

三、在OECM-1細胞中ANE不能誘發EP4的表現........    29

 

四、在OECM-1細胞中PGE2可誘發EMT相關型態.......    29

 

五、COX-2/PGE2不參與OECM-1細胞fibronectin的表現...    31

 

六、在OC3細胞中ANE可藉由COX-2誘發EMT相關表型....    32

 

七、在OC3細胞中ANE可誘發PGE2的分泌..........    33

 

八、在OC3細胞中ANE能誘發EP4的表現..........    33

 

九、在OC3細胞中PGE2可誘發EMT相關型態.........    34

 

柒、討論.......................    36

   
 

捌、圖........................    42

 

圖一、    OECM-1細胞株處理NS-398抑制劑後對vimentin蛋白表現的影響......................... 42

 

圖二、    利用PGE2 EIA kit分析OECM-1細胞株處理ANE後所分泌PGE2的量.......................    43

 

圖三、    OECM-1細胞株處理PGE2後對EP4蛋白表現的影響.. 44

 

圖四、    OECM-1細胞株處理PGE2後對細胞型態的影響....45

 

圖五、    OECM-1細胞株處理PGE2後對vimentin蛋白表現的影響46

 

圖六、    OECM-1細胞株處理PGE2後對細胞型態及vimentin蛋白表現的影響........................ 47

 

圖七、    OECM-1細胞株處理PGE2後對fibronectin蛋白表現的影響.......................... 48

 

圖八、    OECM-1細胞株處理PGE2後對細胞型態及fibronectin蛋白表現的影響...................... 49

 

圖九、    OECM-1細胞株處理NS-398抑制劑後對fibronectin蛋白表現的影響....................... 50

 

圖十、    OC3細胞株處理NS-398抑制劑後對vimentin蛋白表現的影響..........................    51

 

圖十一、    利用PGE2 EIA kit分析OC3細胞株處理ANE後所分泌PGE2的量........................    52

 

圖十二、    OC3細胞株處理PGE2後對EP4蛋白表現的影響....53

 

圖十三、    OC3細胞株處理PGE2後對細胞型態的影響..... 54

 

圖十四、    OC3細胞株處理PGE2後對vimentin蛋白表現的影響. 55

 

玖、圖........................ 56

 

附圖一、    Arachidonic acid代謝圖............56

 

附圖二、    OECM-1細胞中,PDTC於不同時間點抑制ANE所誘發COX-2表現的情形..................    57

 

附圖三、    OECM-1細胞處理ANE後對vimentin蛋白表現的影響. 57

 

附圖四、    OECM-1細胞處理ANE後型態的改變........ 58

 

附圖五、    OECM-1細胞處理ANE後以共軛焦顯微鏡觀察型態及vimentin蛋白表現..................    58

 

附圖六、    OECM-1細胞處理不同濃度的ANEfibronectinvimentin蛋白表現的影響..........................    59

 

附圖七、    OC3細胞處理不同濃度的ANEvimentin蛋白表現的影響.........................    59

 

附圖八、    OECM-1細胞中,ANE於不同時間點對NF-κB活性的調控.........................    60

 

拾、附表....................... 61

 

拾壹、參考文獻....................    64