本實驗室先前以石墨烯氧化物(graphene oxide, GO)結合化療藥物cisplatin (CDDP)用於癌症治療，發現GO結合CDDP (GO/CDDP)可以降低CT26大腸癌細胞對於CDDP之抗藥性，進而提升化療效果。GO/CDDP會引發CT26的細胞自噬(autophagy)、改變CT26之自噬流(autophagic flux)、引發自噬標記蛋白LC3與CDDP的核傳輸(nuclear transport)現象並造成大量細胞壞死。本研究發現GO/CDDP在不同癌細胞(A549、Tramp-C1、HeLa以及Skov-3)會造成不同程度的毒殺效果、自噬、核傳輸現象，以及不同程度的癌細胞壞死。此外，我們以免疫螢光染色證實GO/CDDP在引發細胞自噬後，會提升tubulin乙醯化程度、使microtubule聚合並提升核傳輸現象，因此促進LC3複合物的進核傳輸。為了瞭解細胞核中LC3 複合物組成，我們以LC/MS/MS進行蛋白質鑑定，分析結果顯示，LC3複合物中含有histone H1/H4，因此，我們推測GO/CDDP會影響histone轉譯後修飾的情形。我們以基因表現分析發現，GO/CDDP會提升乙醯化酵素(HAT)及抑制去乙醯化酵素(HDAC) 之表現並造成H4K16ac增加，而在抑制乙醯化/去乙醯化酵素活性並以GO/CDDP處理後，核傳輸現象未受到影響，但從細胞生長狀況發現抑制去乙醯化酵素後，以GO/CDDP處理後之細胞死亡情形大幅提升，我們推測H4K16為此機轉中細胞死亡下游調控蛋白。藉由提升細胞自噬、促進核傳輸現象並造成細胞壞死，GO在具有抗藥性或細胞凋亡卻失的癌細胞中可以作為CDDP之化療增敏劑，因此本研究證實GO/CDDP為具有潛力之化學治療手段。

We previously demonstrated that GO/CDDP could decrease the drug resistance and increase the cytotoxicity in mouse colon cancer cell line (CT26). Our previous study showed that GO/CDDP triggered cell autophagy, elevated nuclear import and induced cell necrosis in CT26. In this study, we found that GO/CDDP caused different degrees of cell cytotoxicity, cell autophagy, nuclear import and necrosis in different cancer cell lines (A549、Tramp-C1、HeLa and Skov-3). Furthermore, the immunostaining showed that GO/CDDP would enhance the acetylation of tubulin which polymerized microtubule and elicited nuclear transport of LC3 complex. To evaluate the LC3 complex in nuclear, we applied LC/MS/MS to proceed protein identification. The results revealed that histone H1/H4 bound to LC3. Therefore, we suggested that GO/CDDP may affect the post-translational modifications. From the results of microarray, GO/CDDP increased the expression level of histone acetyltransferase (HAT) but decreased the expression level of histone deacetylase (HDAC). In addition, GO/CDDP enhanced H4K16ac level. When treated with inhibitors (HATi and HDACi), it did not affect the phenomenon of nuclear transport. But from the observation of cell morphology, pre-treated with HDACi would lead to severe cell death after GO/CDDP treatment. We suggested that histone acetylation was in the downstream of the regulation by GO/CDDP. With the enhancement of autophagy, nuclear import and cell necrosis, GO can be a chemosensitizer of CDDP in drug-resistant/apoptosis-deficient cancer for chemotherapy.

致謝 I
摘要 II
Abstract III
目錄 IV
圖表目錄 VI
第一章 文獻回顧 1
1-1 氧化石墨烯 (Graphene Oxide, GO) 1
1-1-1 GO之介紹 1
1-1-2 GO與奈米碳管(Carbon Nanotubes, CNTs)之比較 1
1-2 細胞自噬 (Autophagy) 1
1-2-1 細胞自噬介紹 2
1-2-2 細胞自噬與組織蛋白(histone)之相關性 3
1-2-3 細胞自噬於癌症上之應用 4
1-3 細胞核傳輸機制介紹 5
1-4 癌症與治療方式 6
1-4-1 癌症介紹 6
1-4-2 癌症治療方式 6
1-5 氧化石墨烯結合化療藥物之應用 7
1-6 實驗動機 8
第二章 實驗材料與方法 17
2-1 細胞培養 17
2-2 實驗材料及化療藥物 17
2-2-1 氧化石墨烯 17
2-2-2 化療藥物 (Cisplatin) 17
2-2-3 氧化石墨烯結合化療藥物(GO/CDDP)處理細胞 17
2-2-4 抑制劑及前處理 18
2-3 細胞毒性測試 (MTT assay) 19
2-4 細胞免疫螢光染色 (Immunofluorescence staining) 20
2-5 螢光顯微鏡分析 22
2-5-1 LC3/p62、LC3/Lamp-2共位現象及LC3 puncta進核定量 22
2-6 核質分離 22
2-7 感應耦合電漿質譜分析 (ICP-MS) 23
2-8 細胞壞死(necrosis)與細胞凋亡(apoptosis)之分析 23
2-9 流式細胞儀 (Flow Cytometry) 24
2-10 西方墨點法蛋白質分析 (Western blot analysis) 24
2-10-1 聚丙烯醯胺膠體電泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 24
2-10-2 西方墨點法 25
2-11 細胞免疫沉澱 (Immunoprecipitation) 27
第三章 實驗結果 28
3-1 GO結合CDDP對不同癌細胞之細胞存活率影響 28
3-2 GO/CDDP對不同癌細胞之細胞自噬流之影響 28
3-3 GO/CDDP對不同癌細胞之核傳輸現象及細胞壞死之影響。 29
3-4 GO/CDDP對不同癌細胞之核傳輸機制影響 30
3-5 利用抑制細胞自噬及核傳輸路徑探討LC3核傳輸現象及細胞壞死情形 31
3-6 利用免疫沉澱探討核內LC3複合物組成情形 32
3-7 Histone相關基因表現 35
3-8 GO/CDDP對histone H1/H4轉譯後修飾之影響 35
3-9 利用histone乙醯化/去乙醯化酵素抑制劑探討GO/CDDP對細胞之影響 36
3-10 利用HATi及HDACi觀察GO/CDDP對histone H4 lysine16之影響 37
3-11 利用HATi及HDACi觀察GO/CDDP對核傳輸之影響 37
3-12 結論 38
第四章 討論與未來展望 51
第五章 參考文獻 54

圖目錄
圖1-1、以電子顯微鏡觀察細胞不同死亡情形。 10
圖1-2、細胞自噬示意圖。 11
圖1-3、細胞自噬相關蛋白調控示意圖。 12
圖1-4、SNAREs參與自噬溶酶體形成之機制。 13
圖1-5、細胞核傳輸機制。 14
圖1-6、105年國人十大死因。 15
圖1-7、105年十大癌症死因。 16
圖3-1 GO結合CDDP對不同癌細胞之細胞存活率影響。 39
圖3-2 GO/CDDP對不同癌細胞之細胞自噬流之影響。 40
圖3-3 GO/CDDP對不同癌細胞之核傳輸現象及細胞壞死之影響。 41
圖3-4 GO/CDDP對不同癌細胞核傳路徑之影響。 42
圖3-5 利用抑制細胞自噬及核傳輸路徑探討LC3核傳輸現象及細胞壞死情形。 43
圖3-6 利用免疫沉澱分離LC3複合物。 44
圖3-7 Histone相關基因表現。 45
圖3-8 GO/CDDP對histone H1/H4轉譯後修飾之影響。 46
圖3-9 利用histone乙醯化/去乙醯化酵素抑制劑探討GO/CDDP對細胞之影響。 47
圖3-10 利用HATi及HDACi觀察GO/CDDP對H4K16ac之影響。 48
圖3-11 利用HATi及HDACi觀察GO/CDDP對核傳輸之影響。 49
圖3-12 GO/CDDP引發癌細胞之機轉。 50

表目錄
表2-1 基因抑制所使用之siRNA及抑制劑 19
表2-2 免疫螢光染色之一級抗體 21
表2-3 免疫螢光染色之二級抗體 22
表2-4 西方墨點法之一級抗體 26
表2-5 西方墨點法之二級抗體 26
表3-1 蛋白質鑑定結果(蛋白質大小約25-35 kDa) 34
表3-2 蛋白質鑑定結果(蛋白質大小約11 kDa) 34

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