論文名稱:

檳榔子萃取液對於中性球分泌趨化激素之影響

 

Effects of areca nut extracts on release of chemokines from neutrophils

研究生:

廖秋菊  Chiu-Chu Liao

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

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:

39217002

          學年度:

93

          語文別:

中文

          出版年:

94

關鍵字:

中性球  neutrophil

 

白質-8  interleukin-8

 

檳榔子萃取液  areca nut extract

 

鈣離子合劑  ANE

 

檳榔嚼塊  EGTA

 

  AQ

全文說明:

(本論文未授權公開)

 

電子全文

        論文頁數:

87

摘要:

嚼食檳榔在東南亞國家是一個非常普遍的習慣,在台灣也有超過兩百萬的人口有嚼食檳榔的習慣。在檳榔與口腔疾病相關的研究顯示嚼食檳榔者比未嚼食檳榔者有較高的牙周病患率。嚼食檳榔者發生口腔黏膜下纖維化症(oral submucous fibrosis)、口腔白斑症(oral leukoplakia)與口腔癌(oral cancer)的機率也比未嚼食檳榔者來的高。先前的研究發現將嗜中性(neutrophils)以檳榔子萃取液(areca nut extractANE)處理後,嗜中性球殺菌、釋放超氧化物及吞噬能力都會下降。對於ANE是否影響中性球分泌細胞激素目前仍不清楚。本研究目的乃探討ANE對人類中性球分泌趨化激素的影響為何,並探討是否受鈣離子調控。

 

嗜中性球乃自健康成人之周邊血純化而得,以不同濃度之ANEHBSS/Ca2+HBSS緩衝液中分別處理8小時後,並利用propidium iodide (PI)染色,以流式細胞儀(flow cytometer)測量中性球的存活度,並收集上清液,利用酵素連結免疫吸附分析法(enzyme-linked immunosorbent assayELISA)分別測量中性球interleukin-8 (IL-8)macrophage inflammatory protein-1α (MIP-1a)之分泌量。進一步,以細胞外鈣離子合劑[ethylene glycol-bis (2-aminoethylether)-N, N, N, N-tetraacetic acid, EGTA]處理中性球1小時之後,再以含6.25μg/ml ANEHBSS/Ca2+緩衝液處理8小時後分別收集上清液測量IL-8MIP-1a,並測量中性球的存活度。

 

    實驗結果顯示溶於HBSS/Ca2+ANE對嗜中性球的存活度影響較溶於HBSS緩衝液下為低。另外,ANE會調控IL-8的分泌,使嗜中性球的IL-8分泌量上升,但不影響MIP-1a的分泌;而不同濃度的ANEHBSS/Ca2+緩衝液下,刺激中性球IL-8的分泌量較在HBSS緩衝液下刺激嗜中性球IL-8的分泌量為高。此外,實驗結果顯示在HBSS/Ca2+緩衝液下,細胞外鈣離子合劑EGTA (2.5 mM)會抑制ANE所調控IL-8的分泌。

 

本研究顯示ANE會刺激中性球分泌IL-8,而且此刺激現象在液體含鈣離子的情形下上升更為明顯;細胞外鈣離子合劑EGTA會抑制ANE所調控IL-8的分泌,而ANE不會影響中性球分泌MIP-1。由此可知檳榔子萃取液影響中性球分泌趨化激素IL-8的功能,可能藉由影響中性球的功能而影響口腔健康。

   
 

Areca quid (AQ) chewing is popular in countries in Southeast Asia. Epidemiological studies have revealed that there are approximately two million AQ chewers in Taiwan. A strong relationship between AQ chewing and oral diseases such as oral cancer, oral leukoplakia, and periodontal disease has been established. Previous studies showed that the exposure of neutrophils to areca nut extract (ANE) reduces the superoxide anion production, the bactericidal and phagocytic activity. However, it is not yet clear whether ANE can affect chemokines secretion by neutrophils. This study aimed to investigate the effects of ANE on chemokines secretion in neutrophils, and to study the potential role of calcium ions.

 

Neutrophils isolated from peripheral blood of healthy volunteers were treated with various concentrations of ANE in HBSS/Ca2+ or HBSS for 8 hours. Afterwards, the effects of ANE on the viability of neutrophils were determined by flow cytometer using propidium iodide (PI) staining. In addition, the cell supernatants after ANE treatment were collected, and the protein levels of interleukin-8 (IL-8) and macrophage inflammatory protein-1 (MIP-1) were analyzed using enzyme-linked immunosorbent assay (ELISA). To study the role of calcium ions, neutrophils were pretreated with extracellular calcium chelator, EGTA [ethylene glycol-bis(2-aminoethylether)-N, N, N’, N’-tetraacetic  acid] for 1 hour followed by treatment of neutrophils with 6.25 慊/ml of ANE in HBSS/Ca2+ for 8 hours. The cell viability was then determined, and the cell supernatants were subjected to IL-8 analysis.

 

The results showed that neutrophils incubated in HBSS were more susceptible to ANE. The IL-8 production, but not MIP-1a production, was induced by ANE in a dose-dependent manner. Moreover, the modulation of IL-8 by ANE was more evident in the presence of calcium ions. In addition, after pretreated with extracellular calcium chelator, EGTA (2.5 mM), the induction of IL-8 by ANE was inhibited.

 

To conclude, the results in this study showed that ANE induced IL-8 secretion of neutrophils, and the calcium ions may play a role in the regulation of ANE-induced IL-8 production. The secretion of MIP-1 by neutrophils was not affected by ANE. The results generated in this study might further elucidate how areca nut influences the functions of neutrophils.