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研究生:林俊甫
研究生(外文):Chun-Fu Lin
論文名稱:L型選擇素(L-selectin,SELL)在口腔癌中過度表現的機制與其功能之探討
論文名稱(外文):An Investigation of the Mechanisms and Functions of L-selectin (SELL) Overexpression in Oral Cancer
指導教授:張國威
指導教授(外文):Kuo-Wei Chang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:75
中文關鍵詞:口腔鱗狀上皮細胞癌L型選擇素癌症轉移
外文關鍵詞:OSCCL-selectinMetastasis
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口腔鱗狀上皮細胞癌(OSCC)為台灣男性第四大癌症,而L型選擇素(L-selectin)屬於選擇素家族的一員,主要表現於白血球細胞膜上,可調控白血球與血管內皮細胞貼附。先前研究顯示在轉移之口腔鱗狀上皮細胞癌組織中,L型選擇素的表現比原位癌組織高,且L型選擇素表現較高之病人存活率也較差,因此認為L型選擇素在癌症轉移的過程中扮演重要的角色。然而其調控機制尚未清楚,因此本研究主要探討L型選擇素在口腔癌中扮演的角色與其調控之機制為何。
首先建構帶有L型選擇素與綠色螢光基因之質體DNA,並藉由活體外實驗顯示過度表現L型選擇素之口腔癌細胞明顯誘發細胞轉移、侵襲與非固著性生長能力,活體實驗則顯示過度表現L型選擇素之SAS細胞明顯誘發腫瘤生長能力,且初步顯示其具有促進轉移至肺部的能力,而L型選擇素之促進子區域在口腔癌細胞中可以誘發L型選擇素表現。根據實驗顯示在口腔癌細胞中過度表現L型選擇素並不會調控MAPK、FAK、Akt、caspase-3、Bcl-2、fibronectin與E-cadherin的表現,但是過度表現L型選擇素之SAS細胞中具有抑制vimentin與cytokeratin表現之結果,因此初步推測在口腔癌細胞中,過度表現L型選擇素或許與抑制細胞骨骼的表現而導致口腔癌細胞分化程度降低,造成口腔癌細胞致癌能力增加有所關連。
Oral squamous cell carcinoma (OSCC) is the fourth most prevalent malignancy in male of our country. L-selectin (SELL) belongs to cell adhesion molecules (CAM), and is one of the selectin family. SELL mainly expresses on the cell membrane of leukocytes, and regulates the rolling and attachment of leukocytes with endothelial cells. However, our previous study showed that the expression of SELL on cells from metastatic lesions were higher than those from primary OSCC. Furthermore, the patients who exhibiting higher SELL expression had lower survival rate than those with lower SELL expression. Therefore, SELL may play a pivotal role in the development of metastasis. Till far, the mechanisms of SELL in promoting neoplastic progression of solid tumor are still unclear. The aims of this study are to investigate the functions of SELL overexpression and the mechanisms regulating SELL expression in OSCC cells.
We constructed the plasmid DNA containing SELL and GFP genes for several phenotypic analysis. The results showed that the SELL-overexpressed OSCC cells had higher abilities of migration, invasion and anchorage-independent growth comparing with control groups in vitro. The in vivo tumorigenic experiments showed significant difference in tumor growth between SELL-overexpressed SAS cells and control groups. We also constructed the plasmid DNA containing the promoter region of SELL gene. The reporter assay showed that the promoter activity of SELL was upregulated about 1.5 fold than control groups. Althogh the SELL-overexpressed SAS cells were assumed to have higher metastatic potential than control groups, the SELL overexpression did not upregulate MAPK, FAK, Akt, apoptosis related (Bcl-2 and caspase-3) or epithelial-mesenchymal transition (EMT) related (fibronectin and E-cadherin) pathways in OSCC cells. However, vimentin and cytokeratin were downregulated in SELL-overexpressed SAS cells. Therefore, SELL overexpression may increase the oncogenic potential in OSCC cells by disrupting cytoskeleton.
目錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥I
中文摘要‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥.V
Abstract‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥VI
圖目錄‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥. VII
壹、緒論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 1
口腔鱗狀上皮細胞癌 …………………………………………1
選擇素家族(Selectin family)……………………………2
L型選擇素的結構與功能………………………………………3
L型選擇素的活化………………………………………………4
L型選擇素的配位體……………………………………………5
L型選擇素與癌症之關係………………………………………6
貳、研究目的‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 7
參、實驗方法與材料‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ 8
一、細胞培養 …………………………………………………8
二、細胞冷凍 …………………………………………………9
三、細胞解凍 …………………………………………………9
四、質體DNA(Plasmid DNA)之製備 ………………………10
五、質體DNA之轉型(Transformation)作用………………13
六、少量質體DNA之製備………………………………………14
七、大量質體DNA之製備………………………………………14
八、病毒之製備 ………………………………………………15
九、病毒感染(Virus Infection)…………………………16
十、西方點墨法(Western Blot) …………………………16
十一、細胞免疫染色(Immunocytochemistry, ICC) ……18
十二、細胞轉染(Transfection) …………………………19
十三、細胞增生實驗(Proliferation Assay)……………20
十四、細胞移行實驗(Migration Assay)…………………20
十五、細胞侵襲實驗(Invasion Assay) …………………21
十六、非固著性生長(Anchorage Independent Growth Assay)………………………………………………………………… 21
十七、腫瘤生長能力(Tumorigenesis Assay)測試………22
十八、轉移能力(Metastasis Assay)測試 ………………22
十九、報導者分析法(Reporter Assay) …………………22
二十、統計分析 ………………………………………………24
肆、結果 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥.25
建構pCDH-SELL-GFP質體並確認其正確性……………………25
建立穩定過度表現L型選擇素的口腔癌細胞株………………25
過度表現L型選擇素並無明顯影響OECM-1細胞之生長能力 …………………………………………………………………26
過度表現L型選擇素誘發口腔癌細胞之爬行能力 …………26
過度表現L型選擇素會誘發口腔癌細胞之侵襲能力 ………27
過度表現L型選擇素會誘發口腔癌細胞非固著性生長能力…28
過度表現L型選擇素會誘發SAS細胞腫瘤生長的能力 ………28
過度表現L型選擇素可能誘發SAS細胞的轉移能力……………29
口腔癌細胞中L型選擇素促進子區域的增加誘發促進子活性…30
TGFβ及IFNβ會些微調升L型選擇素促進子活性 ………………31
Akt的活性不會影響L型選擇素的表現…………………………31
過度表現L型選擇素在口腔癌細胞中所調控的訊息傳遞路徑…32
伍、討論‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥.34
L型選擇素對口腔癌細胞生長能力的影響…………………… 34
L型選擇素對於口腔癌細胞移動與轉移能力的影響 …………35
L型選擇素之促進子區域調控L型選擇素表現…………………35
調控L型選擇素表現之機制探討 ………………………………36
過度表現L型選擇素所調控之下游訊息傳遞路徑 ……………38
陸、圖表‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥41
柒、參考文獻‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥64
捌、附圖‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥69
玖、附表‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥73
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