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研究生:王培紋
研究生(外文):Pei-Wen Wang
論文名稱:微型核醣核酸181於口腔鳞狀上皮癌細胞之角色
論文名稱(外文):The roles of miR-181 in oral squamous cell carcinorma cells
指導教授:林姝君林姝君引用關係
指導教授(外文):Shu-Chun Lin
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:80
中文關鍵詞:口腔鱗狀上皮細胞癌微型核醣核酸181
外文關鍵詞:OSCCmiR-181
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口腔鱗狀上皮細胞癌 ( OSCC ) 為我國非常盛行之癌症,並以男性居多。若能及早診斷,了解其病變過程及基因變化,對於控制此一疾病將極有助益。微型RNA ( microRNAs; miRNAs )由20~23個核苷酸組成,是一群不會轉譯出蛋白質的RNA 。這些RNA經由與標靶基因mRNA的序列配對,可以抑制標靶基因蛋白質的生成,調控其表現。因此miRNAs失調可能導致許多癌症之生成。根據實驗室先前研究發現miR-181在高度轉移的口腔鱗狀上皮細胞癌組織中,表現量比低度轉移或不轉移的口腔鱗狀上皮組織還要高,而表現較多miR-181a和miR-181b的口腔癌病患,術後存活率較低,而miR-181在口腔鱗狀上皮細胞癌之生成及進行角色未明。本實驗研究目的為探討miR-181在OSCC細胞株中的功能,並進一步分析以血液中之miR-181為診斷口腔癌的生物指標之可行性。研究發現,miR-181過度表現會造成細胞生長變慢,移行能力增加,過度表現miR-181也使得fibronectin蛋白質的表現量增加。推測miR-181可能是藉由fibronectin的表現量增加,進而增加細胞的移行能力,使癌細胞能夠轉移到身體其他部位,導致術後存活率較低。在口腔癌病患血漿中miR-181a及miR-181b的表現量與一般健康人比較,miR-181a的表現量較高,因此血液之miR-181a具有作為口腔癌診斷指標之潛力。
Oral squamous cell carcinoma ( OSCC ) is a very prevalent neoplasms in Taiwan, particularly for male population. Early diagnosis and better understanding of the pathogenetic basis of these tumors will bestow profound benefits in treatment of this disease. MicroRNAs ( miRNAs ) are 20~23 nucleotide noncoding RNAs. miRNAs regulate gene expression by base-pairing to the target mRNA for mRNA cleavage or repression of protein translation. Therefore, dysregulation of miRNAs may lead to carcinoma pathogenesis and progression. Previous study in our laboratory showed that the expression levels of miR-181 were higher in highly metastatic OSCC than that in the low metastatic potential or non-metastatic OSCC. Tumor with higher miR-181 express had worse survival rate. However, the roles of miR-181 in the OSCC initiation and progression are unclear. The purposes of this study are to elucidate the roles of miR-181s in OSCC cell lines and to investigate the possibility of miR-181s as a diagnostic biomarker. Overexpression of miR-181 down-regulated SAS cells proliferation, and up-regulate SAS and OC3 cells migration. miR-181 overexpression also up-regulates fibronectin expression. Therefore, it is postulated that miR-181 overexpression may up-regulate cell migration via fibronectin expression to promote cancer cells metastasis in high results in a lower survival. Expression level of miR-181a is higher in the plasma of cancer patients comparing to healthy individuals. These results suggest the potential of plasma miR-181a as a diagnostic marker.
中文摘要 4
Abstract 5
壹、緒論 7
一、口腔癌 7
二、微型RNA 8
三、微型RNA與癌症 11
四、miR-181 12
五、miR-181在臨床口腔癌組織中的表現 14
六、纖維連結蛋白 ( Fibronectin ) 15
七、微型RNA在血液中的偵測 16
貳、研究目標 18
叁、實驗材料與方法 19
一、細胞繼代培養 ( Cell subculture ) 19
二、病毒感染 ( Virus infection ) 21
三、RNA萃取 ( Extraction of RNA ) 21
四、微型RNA的反轉錄 ( Revers transcription ) 及定量聚合酶連鎖反應 ( Real-time PCR ) 22
五、報導基因分析 ( Reporter gene assay ) 23
六、細胞增生分析 ( Proliferation assay ) 25
七、細胞移行分析 ( Migration assay ) 25
八、細胞非貼附性生長分析 ( Anchorage-independence assay ) 26
九、西方墨點法 ( Western blot ) 27
十、統計 29
肆、研究結果 30
一、建立過度表現miR-181之口腔鳞狀上皮癌細胞株 30
二、過度表現miR-181之口腔鳞狀上皮癌細胞株表型分析 31
三、miR-181下游基因 33
四、miR-181於血漿中的表現 36
伍、討論 38
陸、圖例 44
圖一、慢病毒感染口腔鳞狀上皮癌細胞株之綠色螢光蛋白表現 44
圖二、定量反轉錄聚合酶連鎖反應檢測慢病毒感染口腔鳞狀上皮癌細胞株之SAS細胞中miR-181含量 46
圖三、定量反轉錄聚合酶連鎖反應檢測慢病毒感染口腔鳞狀上皮癌細胞株之OC3細胞中miR-181含量 47
圖四、檢測SAS細胞內過度表現的miR-181之功能 48
圖五、過度表現miR-181對口腔鳞狀上皮癌細胞株SAS生長的影響 49
圖六、過度表現miR-181對口腔鳞狀上皮癌細胞株OC3生長的影響 50
圖七、過度表現miR-181對口腔鳞狀上皮癌細胞株移行的影響 51
圖八、過度表現miR-181對口腔鳞狀上皮癌細胞株SAS非貼附性生長的影響 52
圖九、預測軟體預測miR-181的標靶基因在過度表現miR-181口腔鳞狀上皮癌細胞株的表現 53
圖十、過度表現miR-181對口腔鳞狀上皮癌細胞株細胞生長訊息傳遞的影響 54
圖十一、過度表現miR-181對口腔鳞狀上皮癌細胞株缺氧誘發訊息傳遞路徑的影響 55
圖十二、過度表現miR-181對口腔鳞狀上皮癌細胞株細胞分化相關訊息路徑之影響 56
圖十三、過度表現miR-181對口腔鳞狀上皮癌細胞株的EMT相關訊息路徑之影響 57
圖十四、口腔癌病患血液相對於健康人血液中的miR-181表現量 58
柒、附圖 60
附圖一、miR-181在口腔癌組織中的表現量 60
附圖二、miR-181在口腔癌組織中的表現量與血管侵襲的關係 62
附圖三、miR-181的表現與口腔癌病患術後存活的關係 63
附圖四、pCMV-Lac-MCS質體圖 64
附圖五、pCMV- Luc質體圖 65
附圖六、承接miR-181基因的慢病毒質體圖 66
附圖七、miR-181在口腔鳞狀上皮癌細胞株中的表現 67
捌、附表 68
附表一、微型RNA序列 68
附表二、建構過度表現miR-181質體所需miR-181序列之引子與產物序列 69
附表三、溶液成分 70
玖、參考文獻 74
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