臺灣博碩士論文加值系統

口腔癌近10年來死亡率急遽的增加，而淋巴結轉移是造成口腔癌預後不佳的原因。最近文獻顯示，p53和轉錄因子Snail,Twist可能扮演腫瘤抑制者的角色。本研究利用免疫化學染色法探討112例口腔癌樣本其p53,侵襲相關分子標記Twist, Snail和E-cadherin表現與臨床意義。另外探討薑黃素抗口腔鱗狀細胞癌細胞株(SCC-25)侵襲的作用，其侵襲相關分子和基質金屬蛋白酶(MMPs)的表現。結果顯示112例口腔鱗狀細胞癌樣本其p53、Snail和Twist的表現量增加，而E-cadherin的表現量降低。口腔鱗狀細胞癌樣本和良性增生樣本比較，表現量增加的p53(P=0.028)、Twist(P=0.000)和表現量降低的E-cadherin(P=0.001)有顯著差異。E-cadherin與p53(P=0.015)、Snail(P=0.049)有顯著差異，而與Twist表現量之間並無顯著差異。P53和Twist與臨床病理參數無顯著相關，只有Snail表現量與腫瘤大小≦2公分(P=0.028)，腫瘤早期(P=0.012)，吸煙習慣(p =0.043)，觀察期間死亡(p=0.041)等臨床病理參數有顯著相關。單變量存活率分析(univariate analysis)與Snail表現量(P=0.050)、E-cadherin表現量(P=0.024)、腫瘤分期(P=0.043)、腫瘤大小(P=0.001)、腫瘤復發(P=0.000)、抽菸(P= 0.001)、嚼食檳榔的習慣(P=0.006)有顯著的差異。然而，多變量分析(Multivariate analysis)運用logistic regression分析結果顯示五年總存活率與Snail的表現量並無顯著的相關性。綜合以上結果發現Snail可能具有作為診斷預後不良的口腔癌早期病人之潛力標記。另外體外試驗方面，口腔鱗狀細胞癌細胞株(SCC-25)分別以不同濃度的薑黃素(2.5~30μΜ)處理24小時，有抑制增殖和群落形成能力的效果，主要的影響為阻斷G2/M phase，並且促使PARP的表現下降，引發口腔癌細胞凋亡。除此之外，薑黃素還具有抗口腔癌侵襲能力，主要透過活化p53的表現影響上皮轉錄因子Twist和Snail表現下降，促使E-cadherin表現增加，阻止口腔癌細胞的上皮間質轉化(EMT)，再透過抑制基質金屬蛋白酶MMP-2和MMP-9的分泌，使癌細胞無法降解細胞外基質和基底膜，進而抑制口癌細胞的侵襲和轉移，薑黃素具有潛力成為用於預防或治療口腔癌侵襲的藥物。

The mortality of oral squamous-cell carcinoma (OSCC) increased in recent years. One of the major prognostic factors for oral squamous-cell carcinoma (OSCC) patients is regional lymph node metastases. Recent studies have shown the transcription factors P53, Snail, Twist may be played a regulate role of tumor suppressor. This study aimed to determine the invasion-related molecular markers of p53, Twist, Snail and E-cadherin expression in OSCC and their clinical significance. We investigated P53, Snail, Twist and E-cadherin expression by immunohistochemistry with oral cancer (n=112) and benign hyperplasia tissue (n=16). In addition, another aim of study was to evaluate the anti-invasive effect of curcumin on expression of invasion associated moleculars and expression of matrix metalloproteinase (MMPs) in oral squamous cell carcinoma cell lines (SCC-25).
The results showed that expression of P53, Twist and Snail shows increased levels in OSCC, whereas E-cadherin shows reduced. A statistically significant higher expression of Twist (P=0.000), P53 (P=0.028) and lower expression of E-cadherin (P=0.001) was found in OSCC samples compare to benign hyperplasia tissue.A statistically significant expression of E-cadherin and p53 (P=0.015), Snail (P=0.049) expression, no statistically significant correlations were found between Twist and E-cadherin expression. The clinicopathogical parameters were not correlated with P53 and Twist expression.Only Snail expression was significantly associated with tumor size ≦ 2 cm (P=0.028), tumor stage≦ Π(P=0.012), smoking habit (P=0.043) and mortality (P=0.041). Survival analysis showed that positive Snail expression (P=0.050) and E-cadherin expression (P=0.004), tumor size (P=0.001), tumor stage (0.043), recurrence (P=0.000), smoking (P=0.001) and betel nut (P=0.006) were poor prognostic factors. However, Multivariate analysis with logistic regression for 5-year overall survival showed positive expression of Snail was not independent prognostic factor in OSCC. Also, positive Snail expression may be play a part early stage progression and poor prognostic factor. It could be a very useful molecular marker of progression in oral cancer. In vitro study, curcumin (2.5~30μΜ), 24-hour treated on oral squamous cell carcinoma cell lines (SCC-25), could inhibit cell proliferation and induce cell apoptosis via blocking the G2/M phase, and decrease expression of PARP protein. In addition, curcumin has anti-invasive effect, the mechanism related to two steps: First, the p53 activated by curcumin could decrease twist-and-snail expression and increase E-cadherin expression, and thus prevent epithelial mesenchymal transition of oral cancer. Second, curcumin could inhibit the matrix metalloproteinase MMP-2 and MMP-9 expression, so that cancer could not degraded extracellular matrix and basement membrane. These two steps thereby inhibit the invasion and metastasis of oral cancer. Curcumin has the potential to become a drug for the prevention or treatment of oral cancer invasion.

致謝 I
中文摘要 II
英文摘要 IV
目 錄 VI
圖 目 錄 IX
表 目 錄 X
第一章 緒 論 1
1.1 口腔癌(Oral cancer) 1
1.2 口腔癌的病理特性 2
1.3 口腔癌的臨床分期 2
1.4 癌侵襲與口腔鱗狀上皮細胞癌 3
1.4.1 基質金屬蛋白酶(Matrix metalloproteases,MMPs) 4
1.4.2 上皮間質轉化(Epithelial-mesenchymal transitions ,EMTs)的分子標記 5
1.5 p53抑癌基因 6
1.6 細胞週期(Cell cycle) 7
1.7 細胞凋亡(Apoptosis) 8
1.8 口腔癌的治療 8
1.9 薑黃素(Curcumin) 9
第二章 研究材料 12
2.1實驗架構 12
2.1.1臨床分析 12
2.1.2實驗樣本 12
2.2.1體外模式 14
2.2.2實驗樣本 14
2.2.3實驗用細胞株 14
第三章 研究方法 15
3.1臨床分析 15
3.1.1免疫組織化學染色(Immunohistochemical stain) 15
3.1.2統計分析 16
3.2癌細胞特性實驗 16
3.2.1 細胞株之解凍 16
3.2.2 細胞株之凍存 17
3.2.3 細胞之繼代培養 17
3.2.4 細胞存活率分析(Trypan blue dye exclusuin assay) 17
3.2.5 細胞群落形成分析(Cell colony formation assay) 18
3.2.6 細胞週期分析(Cell cycle analysis) 18
3.2.7 細胞凋亡實驗(Apoptosis assay) 19
3.2.8 細胞侵襲能力實驗(Invasion assay) 20
3.3免疫轉漬法 20
3.3.1細胞蛋白質萃取 20
3.3.2蛋白質濃度測定 21
3.3.3 SDS-聚丙烯醯胺膠體電泳(SDS-Polycarylamide Gel Electrophoresis) 21
3.3.4蛋白質轉漬 22
3.3.5免疫轉漬法(Immunoblot) 23
第四章 結果 25
4.1臨床分析之結果 25
4.1.1 112例口腔鱗狀細胞癌其臨床病理特徵 25
4.1.2 P53基因，Snail，Twist與E-cadherin在口腔鱗狀細胞癌的表現 25
4.1.3 112例口腔鱗狀細胞癌其P53，Snail，Twist，E-cadherin與臨床病理特徵之相關性 26
4.1.4 112例口腔鱗狀細胞癌其P53，Snail，Twist，E-cadherin，臨床病理特徵之整體存活率分析 27
4.2薑黃素對口腔癌細胞之分子機制 28
4.2.1薑黃素對口腔癌細胞生長之抑制 28
4.2.2薑黃素對口腔癌細胞的群落形成能力之影響 28
4.2.3薑黃素對口腔癌細胞之細胞週期影響與細胞週期相關蛋白表現量之變化 29
4.2.4薑黃素誘導口腔癌細胞凋亡之分析與細胞凋亡相關蛋白表現量之變化 29
4.2.5薑黃素對口腔癌細胞侵襲抑制之影響與侵襲相關蛋白表現量之變化 30
第五章 討論 32
5.1臨床結果分析 32
5.2薑黃素對口腔癌細胞之分子機制 33
第六章 結論 37
圖與表格說明 38
參考文獻 57

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二、中文部分
行政院衛生署，(2012)，行政院衛生署衛生統計資訊網。
民國97年癌症登記報告，(2011)，行政院衛生署國民健康局統計資訊網。
民國98年癌症登記報告，(2012)，行政院衛生署國民健康局統計資訊網。