論文名稱:

口腔癌基因甲基化異常之鑑定

 

Identification of DNA with abnormal methylation in oral squamous cell carcinomas using methylation sensitive restriction fingerprinting

研究生:

柯順耀  Shu-Yao Ke

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

林姝君  Shu-Chun Lin

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

860H03

          學年度:

87

          語文別:

中文

          出版年:

88

關鍵字:

口腔鱗狀上皮癌  Squamous Cell Carcinoma

 

甲基化  Methylation

 

甲基化限制酵素敏感性指紋鑑定  Methylation Sensitive Restriction Fingerprinting

        論文頁數:

59

摘要:

基因甲基化異常是腫瘤細胞常見的現象,這些變異常在基因促進區﹙promoter region﹚之CpG island上發生,而造成轉錄失活或是突變。近年來一種以基因擴大術為基礎的方法,稱之為甲基化限制酵素敏感性指紋鑑定﹙全名︰Methylation-Sensitive Restriction Fingerprinting,簡稱MSRF﹚,被引用從腫瘤中分離甲基化異常的基因。在此研究使用MSRF鑑定口腔鱗狀上皮癌中甲基化異常的基因。先以在促進區罕見限制位置的限制酵素Mse IDNA切碎,再使用對CpG island甲基化敏感之限制酵素Bst UI處理之後,將尾端帶有Bst UI切點的primer任意組序作基因擴大,目前已經找到48個有改變的DNA,其中包括3個甲基化過量、14個甲基化降低、22個刪除和9個擴大。將這些DNA片段植入質體加以純化,經讀序後和人體基因庫比對,以瞭解其為已知或是未知的基因。其中有一暫時稱為5-3 DNA,其序列和C-terminal binding protein 2 (CtBP2)相似,進一步證明此DNA存在於正常細胞中,且可以RT-PCR的方式在部分癌細胞株測得其表現。

   
 

Abnomal DNA methylation is usually observed in tumor cells. These changes often clustered in CpG island of promoter region of genes. That results in loss of transcription or mutation. Recently, a PCR-based method, named methylation-sensitive restriction fingerprinting (MSRF) has been used to identify rrantly DNA methylation in tumors. In this study, genes with abnormal methylation were identified in oral squemous cell carcinomas (OSCCs) using MSRF. Genomic DNA was first digested by MseI restriction enzyme because its restriction site rarely occurs in promoter region, then treated with a methylation sensitive restriction enzyme Bst UI. Used arbitrary primers to amplify DNA, forty-eight DNA changes in OSCC were found, which includes three hypermethylation, fourteen hypomethylation,twenty-two deletion and nine amplification. Subsequent sequencing and matching to database revealed the identify of these DNA. One of the DNA fragments, named 5-3, which shows high homology to C-terminal binding protein 2 (CtBP2). It was found that 5-3 DNA exists in normal genomic DNA and is expressed in several cancer cell lines.