臺灣博碩士論文加值系統

雖然檳榔子 (Areca Nut) 已被證明與口腔中各種病變有密切的關係，但是致病之分子機轉仍不是很清楚。本研究以口腔癌細胞株OC2, OCSL和 SAS為材料，發現在無血清狀態下，以檳榔子萃取物處理這些口腔癌細胞株會引起細胞強烈的腫脹，以DAPI染色法證實細胞腫脹的同時也引起細胞核顯著的固縮，所觀察到的型態就接近臨床上檳榔嚼食者的病理組織，顆粒層細胞膨脹與基底層細胞核收縮。 以EtBr/AO、PI染色法、染色體完整性分析等方法驗證，證實此腫脹是屬於一種新型細胞壞死，我們稱之核固縮型壞死。以各種抑制劑處理細胞，發現檳榔子萃取液誘導的核固縮型壞死是經由ROS與鈣離子所造成。此外，抑制GSK3β可以加速此核固縮型壞死。補充血清成份之一胰島素可以舒緩此核固縮型壞死。有趣的是，研究的模式中處理檳榔子萃取液會造成顯著的褐色物質累積，這與檳榔嚼食患者的臨床病理症狀，褐色物質覆蓋在上皮細胞亦相符。總結而言，我們建立了一個細胞培養模式，更相似於嚼食檳榔患者的臨床病理症狀，並使用此模式探討檳榔嚼食者的口腔黏膜之脹大和核固縮，未來可以進一步探討檳榔是如何引起慢性發炎反應，而導致腫瘤的發展，乃至於轉移之機制。

Although areca nut has been implicated in various pathogenic effects in oral cavity, the molecular mechanism is still illusive. In this study, oral cancer cells OC2, OCSL and SAS were used as the cell model to investigate areca nu-associated oral pathogenesis. Under serum-free condition, these oral cancer cells treated with areca nut extract (ANE) caused strong cell ballooning. DAPI staining confirmed that this cell swelling concomitantly resulted in significant pyknosis, the morphological alterations frequently observed in betel quid chewers DAPI staining, ethidium bromide/acridine orange double staining, PI staining and DNA fragmentation assay confirmed that these ANE-treated cells a novel state of necrosis, called pyknotic necrosis. Using various inhibitors, we demonstrated that ANE-induced pyknotic necrosis was mediated through reactive oxygen species (ROS) and calcium flux. Besides, GSK3beta inactivation exacerbated ANE-induced pyknotic necrosis. Supplement of insulin was capable of alleviating the ANE-induced pyknotic necrosis. Interestingly, ANE treatment resulted in the accumulation of brown pigment under serum-free condition, resembling clinical and pathological symptoms observed in patients of betel quid chewers. Taken together, we have mimic clinical and pathological conditions in betel quid chewers. In the future, we will take advantage of this model to decipher the progress from initiation of ANE-induced chronic inflammation, followed by pathogenesis, and eventually to tumorigenesis and even metastasis.

目錄
中文摘要 1
Abstract 2
目錄 3
圖目錄 5
第一章 介紹 6
1. 口腔癌 6
2. 口腔癌前病變 7
3. 檳榔 8
4. 檳榔與口腔癌前病變 8
5. 細胞的死亡 9
5.1壞死 9
5.1.1鈣離子(calcium)與壞死 10
5.1.2活性氧化物(reactive oxygen species, ROS)與壞死 11
5.2凋亡(apoptosis) 11
5.3自噬(autophagy) 12
6. 糖原合成酶激酶-3(glycogen synthase kinase-3, GSK3) 12
7. 血清(serum) 13
8. 研究動機 14
第二章 材料與方法 15
1. 材料 15
1.1 細胞株 15
1.1.1 OC2和OCSL 15
1.1.2 SAS 15
1.1.3 HaCaT 15
1.2 培養基 16
1.2.1 DMEM (Dulbecco’s Modified Eagle Medium) 16
1.2.2 RPMI-1640 16
1.3 藥品 17
1.3.1 細胞培養相關之藥品 17
1.3.2 一般實驗上使用 17
1.3.3 緩衝液與配方 19
1.3.4 實驗儀器 21
2. 方法 22
2.1 檳榔萃取液 (ANE) 的製備 22
2.2 細胞培養 22
2.2.1 OC2與OCSL 22
2.2.2 HaCaT與SAS 22
2.3 免疫螢光 22
2.3.1細胞核染色與定量 22
2.3.2 細胞死亡評估 23
2.4 染色質完整性分析 (DNA fragmentation assay) 24
2.5 細胞內活性氧 (Reactive Oxygen Species, ROS)濃度測定 25
2.6 細胞內鈣離子濃度測定 25
2.7 細胞褐色物質沉澱觀察 26
2.8 統計分析 28
第三章 結果 29
第四章 討論 38
第五章 參考文獻 45
第六章 表與圖 52
第七章 附錄 78

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