論文名稱:

檳榔子萃取液對於人類嗜中性白血球分泌前列腺素E2、白細

 

胞介素- 8以及訊息傳遞途徑活性之影響

 

Effects of areca nut extract on release of prostaglandin

 

E2 , interleukin-8 and signaling pathway activity in

 

neutrophils

研究生:

張慧文  Hui-Wen Chang

指導教授:

洪善鈴  Shan-Ling Hung

        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:

39417001

          學年度:

95

          語文別:

中文

          出版年:

96

關鍵字:

嗜中性白血球  neutrophil

 

檳榔子萃取液  areca nut extract

 

前列腺素 E2  prostaglandin E2

 

白細胞介素-8  interleukin-8

全文說明:

(本論文 20100828 對校內公開)

        論文頁數:

80

摘要:

    嚼食檳榔與牙周病(periodontal disease)、口腔白斑症 (oral leukoplakia)和口腔癌 (oral cancer)等口腔疾病具有相關性。嗜中性白血球(polymorphonuclear neutrophils, PMNs)會經由血液進入牙齦溝液(gingival crevicular fluid, GCF)執行非專一性防禦。花生四烯酸(arachidonic acid)經過環氧合酶 (cyclooxygenase, COX)作用生成前列腺素(prostaglandins, PGs)。被活化的嗜中性白血球會釋放細胞激素(cytokines),其中白細胞介素-8 (interleukin-8, IL-8)為化學趨化因子(chemotactic factor)。研究顯示檳榔子萃取液(areca nut extract,ANE)在含有鈣離子的溶液中,會刺激嗜中性白血球分泌prostaglandin E2 (PGE2)IL-8,可以活化嗜中性白血球p38 MAPK (mitogen-activated protein kinase)訊息傳遞途徑,ANE也會造成細胞內鈣離子的上升。本研究目的為探討ANE對發炎因子包括PGE2IL-8的影響,以及細胞內鈣離子、MAPKCOX-2途徑在其中可能扮演之角色。

 

    將健康成人之周邊血液純化而得的嗜中性白血球,以不同濃度ANE,分別在含鈣(HBSS/Ca2+)或不含鈣(HBSS)的緩衝液中處理8小時。利用propidium iodide (PI)染色及流式細胞儀測定顆粒性、細胞大小及細胞存活度的改變。利用免疫方法(immunoassay)偵測PGE2IL-8的釋放情形,進一步了解ANE在含鈣或不含鈣的緩衝液中對嗜中性白血球的影響。並且利用細胞內鈣離子螯合劑(BAPTA-AM)p38ERK MAPKCOX-2的抑制劑,觀察嗜中性白血球PGE2的釋放和IL-8的分泌是否受到調控。

 

    研究結果發現以ANE處理嗜中性白血球8小時,在含鈣的緩衝液之中,隨著ANE處理濃度升高而PGE2IL-8分泌上升,此外,BAPTA-AM會抑制ANE所引起嗜中性白血球PGE2IL-8產生的現象。p38 抑制劑(SB203580)ERK抑制劑(U0126)COX-2抑制劑(NS398)皆會抑制ANE所引起嗜中性白血球PGE2IL-8產生的現象。

 

    本論文研究證明ANE會造成嗜中性白血球PGE2的釋放和IL-8的分泌,而鈣離子的存在具有調控的作用,並且顯示ANE可能藉由增加細胞內鈣離子濃度而增加嗜中性白血球PGE2IL-8分泌的現象,此外,ANE所引起嗜中性白血球PGE2IL-8產生的現象,與p38ERK MAPK的訊息傳遞路徑有關。由此可知ANE能夠刺激嗜中性白血球PGE2的釋放及IL-8的分泌,可能藉由調控嗜中性白血球的功能而影響口腔健康。

   
 

  Areca quid (AQ) chewing is associated with many oral diseases, such as periodontal disease, oral  leukoplakia, and oral cancer. Polymorphonuclear neutrophils (PMNs) can migrate from blood to gingival crevicular fluid (GCF) and execute non-specific defense. Prostaglandins (PGs) are synthesized via the cyclooxygenase (COX) pathway. Interleukin-8 (IL-8) is a cytokine and chemotactic factor secreted by activated PMNs. Areca nut extract (ANE), the main component of AQ, enhances the phosphorylation of p38 MAPK (mitogen-activated protein kinase) and intracellular calcium.

 

    The purpose of the study was to investigate the effects of ANE on the production of prostaglandin E2 (PGE2) and IL-8 by PMNs, and to study the potential role of intracellular calcium ions, MAPK and COX-2 pathway involved in the effects of ANE.

 

    PMNs isolated from peripheral blood of healthy volunteers were treated with various concentrations of ANE in HBSS/Ca2+ or HBSS for 8 hours. The effects of ANE on granularity, size, and the viability of PMNs were determined by flow cytometer after propidium iodide (PI) staining. In addition, the cell supernatants after ANE treatment were collected. The amounts of PGE2 and IL-8 in culture supernatants were messured using immunoassays. The effects of intracellular calcium chelator (BAPTA-AM), the p38 MAPK inhibitor (SB203580), the ERK MAPK inhibitor (U0126), and the COX-2 inhibitor (NS398) were also examined.

 

The results showed that ANE significantly increased the secretion of PGE2 and IL-8 by PMNs in HBSS/Ca2+. In addition, after pretreated with the intracellular calcium chelator, the p38 MAPK inhibitor, the ERK MAPK inhibitor, and the COX-2 inhibitor, the inductions of PGE2 and IL-8 by ANE were inhibited. 

 

    In conclusion, the results in this study showed that ANE induced secretion of PGE2 and IL-8 by PMNs, The inducible effects of ANE may be due to the increase of the intracellular calcium. Moreover, p38 and ERK MAPK may play a role in the regulation of ANE-induced PGE2 and IL-8 production. The results might help to explain the influences of AQ chewing on oral health.

   

        論文目次:

中文摘要………………………………………………………...I

 

英文摘要…...……………………………………………………III

 

緒論……….……………………………………………………..1

 

材料與方法……...………………………………………………18

 

結果……...………………………………………………………31

 

討論………...……………………………………………………44

 

附圖………...……………………………………………………50

 

附錄………...……………………………………………………70

 

參考文獻……...…………………………………………………71