臺灣博碩士論文加值系統

我們過去發現檳榔子（areca nut, AN）所榨出的汁液（AN juice, ANJ）以及其分子量介於30-100 kDa的部分（ANJ 30-100K）均具有誘導細胞進行自體吞噬（autophagy）死亡的活性。目前已知ANJ 30-100K誘導自體吞噬作用需仰賴活潑氧族、Beclin 1與Atg5。本研究擬探討胞吞作用（endocytosis）是否亦為ANJ 30-100K作用之所需；此外，我們也試圖進一步純化ANJ 30-100K。透過化學抑制劑與shRNA等方法，我們證實了格形蛋白（clathrin）與窖蛋白（caveolin）所媒介的胞吞作用，在口腔癌OECM-1與食道癌CE81T/VGH細胞中，均參與了ANJ 30-100K所誘導的細胞毒性與自體吞噬。另一方面，我們利用等電位聚焦法，將分離過ANJ 30-100K的膠條切成十段，並將其中的內含物溶於培養基中。結果顯示其中的第二、四、七與九等四個膠條片段，其溶出物具有細胞毒性，其中以第七段的活性－刺激細胞漲大、細胞質內的物質被大量分解與細胞核皺縮等外觀變化（與ANJ 30-100K所刺激的細胞外觀變化類似），最為明顯。此外，這四段的溶出物均具有誘導LC3-II堆積的活性，但以第七段與第九段膠條最顯著，未來將繼續分析其分子組成。

We previously found that areca-nut- juice (ANJ) and its 30-100 kDa fraction (ANJ 30-100K) are endowed with the activity to induce autophagic cell death. ANJ 30-100K- induced autophagy is currently known to rely on active oxygen species, Beclin 1, and Atg5. This study was to analyze whether endocytosis is required for the activity of ANJ 30-100K. Furthermore, we also tried to further purify the components that induce autophagy in ANJ 30-100K. By using chemical inhibitors and short hairpin RNAs, we demonstrated that both clathrin- and caveolin-mediated endocytosis are required for ANJ 30-100K-mediated cytotoxicity and autophagy. On the other hand, ANJ 30-100K was subjected to separation by isoelectric focusing and the gel strip was dissected into 10 sections and dissolved the containing materials in culture medium. The results showed that materials contained in sections 2, 4, 7, and 9 are cytotoxic. Among them, molecules from section 7 evidently induced swollen cell morphology, massive degradation of cytoplasmic constituents, and shrunked nucleus－similar to morphologic changes induced by ANJ 30-100K. Meanwhile, materials dissolved from the four sections elevated LC3-II levels, and such inducing effects were more profound with those from sections seven and nine. The molecular compositions of molecules contained in section 7 and 9 will be further analyzed.

目錄
中文摘要 I
Abstract II
目錄 III
縮寫表 V
第一章 緒論 1
1-1檳榔嚼塊介紹： 1
1-2 嚼食檳榔及口腔癌之間的相關性： 2
1-3細胞的死亡方式： 3
1-4 自體吞噬（autophagy）： 3
1-5 ANJ 30-100K的發現： 4
1-6 胞吞作用（endocytosis）： 5
1-7 Methyl-β-cyclodextrin（MβCD）與窖蛋白媒介的胞吞作用： 6
1-8胞吞作用與自體吞噬相關性： 7
1-9等電位聚焦電泳（IEF）： 8
1-10 研究動機： 8
第二章 材料與方法 10
2-1 儀器與試劑 10
2-1-1 儀器 10
2-1-2 試劑 11
2-2 實驗方法 16
2-2-1 細胞株的種類及來源 16
2-2-2 細胞培養方法 16
2-2-3 檳榔子萃取液（ANJ 30-100K）的製備 17
2-2-4 細胞存活率分析（Cell viability assay） 17
2-2-5 西方墨點法（Western blot） 18
2-2-5.1 胞液的收集（collection of cell lysate） 18
2-2-5.2 蛋白質濃度測定( protein assay ) 19
2-2-5.3 電泳及轉漬 19
2-2-6 轉染作用 (Transfectation) 21
2-2-6.1 ShRNA的製備 21
2-2-6.2 質體送入細胞內 22
2-2-7 IEF（1D） 23
2-2-8 膠體製程樣品 24
第三章 結果 26
3-1抑制Dynamin的表現對ANJ 30-100K誘導的細胞毒性與自體吞噬之影響 26
3-2抑制格形蛋白的表現對ANJ 30-100K誘導的細胞毒性與自體吞噬之影響 26
3-3 methyl-β-cyclodextrin對ANJ 30-100K誘導的細胞毒性與自體吞噬之影響 27
3-4 窖蛋白的shRNA對ANJ 30-100K的細胞毒性與自體吞噬之影響 28
3-5 ANJ 30-100K的純化 29
第四章 討論 30
第五章 結論 32
參考文獻 33
附表與附圖 40

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