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研究生:曹嘉芬
研究生(外文):Chia-Fen Taso
論文名稱:胃部瀰漫性大型B細胞淋巴瘤之染色體及基因探討
論文名稱(外文):Genomic Investigation of Gastric Diffuse Large B-cell Lymphoma
指導教授:翁文慧
口試委員:蕭崇德馮思中
口試日期:2012-06-21
學位類別:碩士
校院名稱:國立臺北科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:53
中文關鍵詞:瀰漫性大型B細胞淋巴瘤螢光原位雜合技術頻譜影像核型分析染色體G條紋分析TNFAIP3BCL2
外文關鍵詞:Diffuse large B-cell lymphoma (DLBCL)uorescence in situ hybridization (FISH)Spectral karyotyping (SKY)G-bandingTNFAIP3BCL2
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研究背景:瀰漫性大型B細胞淋巴瘤在非何傑金氏淋巴瘤中佔30~40%比例,主要好發於成年人。根據Rosenwald A et al. 曾以微陣列晶片分析基因之表現模式,將DLBCL分為三個亞型型態:Germinal center B cell-like (GCB)、activated B cell-like (ABC)以及primary mediastinal B cell lymphoma (PMBL),配合其臨床資料發現病人均表現不同的預後結果。研究動機:過去研究結果證明,核轉錄因子-κB 在淋巴瘤中扮演著重要的角色,相關訊息傳導途徑中之基因TNFAIP3及基因BCL2 皆是影響的重要因子之一。此外,染色體易位t(14;18)在瀰漫性大型B細胞淋巴瘤中發生頻繁。假設:由此我們可以得知在淋巴癌中,這兩項重點發現應在此疾病中佔有一定的研究價值。實驗材料與方法:本實驗共收集二十二個臨床個案石蠟包埋樣本及一株新建立的瀰漫性大型B細胞淋巴瘤細胞株。我們利用螢光原位雜合技術探討基因TNFAIP3及基因BCL2的表現;另外再利用頻譜影像核型分析法及染色體G條紋分析法探討細胞株染色體之變異性。實驗結果:基因TNFAIP3在個案中普遍缺失一個基因拷貝數(51%);此外,基因BCL2也是存有相同現象(41%),少數個案呈現基因分裂(18%)。細胞株染色體的分析發現,t(5;10)(q22;q24), t(8;22)(q24;q11)以及染色體18號缺失和染色體19號的缺失為此細胞株之主要變異特徵。結論:基因TNFAIP3、BCL2的缺失與染色體t(5;10)(q22;q24), t(8;22)(q24;q11)易位均可能在瀰漫性大型B細胞淋巴瘤發生過程中扮演重要的角色,其為來可能做為此腫瘤之重要生物標記之一。

Background: Diffuse large B-cell lymphoma (DLBCL) is the most intermittently occurred lymphoid malignancy, especially in adulthood which represents 30%–40% of all non-Hodgkin’s lymphoma (NHL). According to a genetic profiling results reported by Rosenwald A et al., DLBL is classified into three major subtypes: germinal center B-cell (GCB), activated B cell-like (ABC) DLBCL and primary mediastinal B cell lymphoma (PMBL). Motivation: Based on the previous studies, constitutive activation of nuclear factor-κB (NF-κB) plays a pivotal role in tumorigenesis of lymphoid malignancies. In addition, TNFAIP3/BCL2 may be responsible for the NF-κB signaling transduction and result in the formation of diffuse large B cell lymphoma. In-spite of other chromosomal aberrations, t(14;18) is the most commonly investigated aberration type in DLBCL. Hypothesize: The alterations of TNFAIP3/ BCL2 or chromosomal translocation (14;18) may responsible for the formation of diffuse large B cell lymphoma. Materials and Methods: In the present study, we have collected twenty-two formalin-fixed paraffin-embedded (FFPE) DLBCL samples, and new established DLBCL cell line. Molecular biology techniques including fluorescence in situ hybridization (FISH) was used to investigated the aberrations of genes TNFAIP3 and BCL2 in all DLBCL samples. Spectral karyotyping (SKY) and G-banding were used to uncover the role of chromosomal aberrations only in the DLBCL cell line. Results: The FISH analysis revealed the one copy number loss in genes TNFAIP3 (51%) and BCL2 (41%), some cases observed gene structure break-apart (18%) in BCL2. The karyotyping results showed t(5;10)(q22;q24), t(8;22)(q24;q11) were the major types of chromosomal translocations in currecnt cell line. Conclusion: Our key observations related to the chromosomal translocations and one copy number loss in genes TNFAIP3 and BCL2 were the crucial genetic abnormalities that may be lead to formation of diffuse large B cell lymphoma, which may further used as biomarkers of DLBCL.

中文摘要 ........II
ABSTRACT........IV
致謝.............VI
表目錄...........IX
圖目錄...........X
第一章 緒論...............1
1、淋巴瘤(Lymphoma)......1
1.1淋巴瘤的現況與分類......1
1.2胃部之瀰漫性大型B細胞淋巴瘤之亞型..7
1.3 常見的胃部淋巴瘤...............8
1.4 NF-κB訊號傳導路徑相關功能.......9
1.4.1核轉錄因子之功能..............9
1.4.2基因 TNFAIP3 之功能..........10
1.4.3基因 BCL2之功能..............10
第二章 研究目標....................13
第三章 材料與方法..................14
3.1 材料.........................14
3.1.1 瀰漫性大型B細胞淋巴瘤病人及腫瘤之樣本....14
3.1.2 瀰漫性大型B細胞淋巴瘤細胞株............14
3.2 實驗方法..............................16
3.2.1 螢光原位雜合技術 ................16
3.2.1.1 原理 ........................16
3.2.1.2 實驗流程 ........................17
3.2.1.2.1福馬林固定石蠟包埋(FFPE)之組織樣本處理....17
3.2.1.2.2 細菌人工染色體 (BAC)萃取與探針 (probe)製備..19
3.2.1.2.3 Nick translation............21
3.2.1.2.4 雜合 ...................22
3.2.2染色體G條紋分析.................24
3.2.2.1 實驗原理...................24
3.2.2.2 實驗流程 .................24
3.2.2.2.1中期染色體之玻片製備........24
3.2.2.2.2染色體染色................25
3.2.3 頻譜影像核型分析.............27
3.2.3.1 原理....................27
3.2.3.2 實驗流程................27
3.2.3.2.1中期染色體之玻片製備.......27
3.2.3.2.2中期染色體之玻片預處理......29
3.2.3.2.3 雜合..............29
3.2.3.2.5 檢測............31
第四章 實驗結果.........33
4.1 以螢光原位雜交法探討NF-κB途徑相關基因之變異....33
4.2細胞遺傳學染色體分析探討胃部瀰漫性大型B細胞淋巴瘤細胞株之結果...40
第五章 實驗討論.....43
參考文獻...48

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