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研究生:林雅琪
研究生(外文):Ya-Chi Lin
論文名稱:台灣地區EB病毒相關之NK/T細胞淋巴癌的分子遺傳研究
論文名稱(外文):Molecular genetic analysis of EBV-associated nasal NK/T cell lymphoma in Taiwan
指導教授:孫孝芳孫孝芳引用關係
指導教授(外文):H. Sunny Sun
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:72
中文關鍵詞:淋巴癌
外文關鍵詞:EBVNK/T cell
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EB病毒相關之NK/T細胞淋巴癌是一特別地好發於東方族群的惡性淋巴癌,其中以鼻腔為最常發病的部位。EB病毒與NK/T細胞淋巴癌之間具有高度的相關性,然而對於EB病毒在NK/T細胞淋巴癌的疾病發生當中所扮演的角色尚無充分地了解,此外對於NK/T細胞淋巴癌腫瘤發生的分子機制所知也十分有限。除了病毒和環境的因素之外,遺傳背景及基因的改變一直被認為是重要的相關因素。近幾年來分子醫學的快速發展使我們瞭解癌症的發生、惡化和轉移的機轉,是由一連串的基因失去調控累積而成,例如致癌基因的過度表現以及抑癌基因的失去活性等,而抑癌基因的功能喪失被認為是可能是癌症發生早期最重要的變化。藉由先前細胞遺傳的研究結果顯示鼻腔區發生之NK/T細胞淋巴癌有高頻率的染色體6q21-q25缺失現象,此區域極可能存有與NK/T細胞淋巴癌相關的重要抑癌基因。在之前其他的癌症研究,藉著聚合脢連鎖反應放大特定的小微星標竿(microsatellite)再利用電泳分析正常細胞及癌細胞內DNA片段的異質性喪失(loss of heterozygosity, LOH),可以成功地找出抑癌基因。此外,對於癌細胞株或是針對只能取到腫瘤組織的狀況,則可以應用同質性定位缺失(homozygosity mapping-of-deletion, HOMOD)的分析方法,而找出抑癌基因的所在位置。在本篇論文當中,為了對於鼻腔區NK/T細胞淋巴癌相關的癌症發生機制能有更進一步的瞭解,利用在染色體6q21-q25區域上的26個具有高度多型性的小微星標竿,進行異質性喪失(LOH)以及同質性定位缺失(HOMOD)兩種分子遺傳學方法分析,期望縮小與NK/T細胞淋巴癌相關的重要抑癌基因所在區域範圍。我們已經完成50個檢體的分析工作,並決定出最小重複範圍(smallest region of overlapping, SRO)的缺失片段約是3.4 Mb。在這個關鍵區域上可能存在與鼻腔區NK/T細胞淋巴癌發生和進展上具有重要影響的基因,相信在不久的將來定能在這些候選基因當中找到與NK/T細胞淋巴癌有關的抑癌基因,以作為往後癌症的臨床診斷之改進以及基因治療之工具。
NK/T cell lymphoma is a new distinct subtype of malignant lymphoma that occurs more prevalently in Asia than in North America and Europe. The nasal region is the most common site of presentation in this tumor and it pursues a highly aggressive course. High incidences of Epstein-Barr virus (EBV) infection in this type of neoplasm have been observed, yet the role of EBV in etiology of the disease is poorly understood. Besides the knowledge on detailed mechanism of tumorigenesis is still very limited. Previous studies have identified a common chromosomal deletion at 6q21-q25 from patients with CD56+ putative NK/T cell lymphoma/leukemia, thus suggesting the region may contain tumor suppressor (TS) genes involved in the carcinogenic process. Loss of heterozygosity (LOH) that may result from deletions or other genetic events is generally used to detect the inactivation of a nearby TS gene. Additionally, the homozygosity mapping-of-deletion (HOMOD) analysis has been developed in adjunction of LOH analysis to assist in localization of TS gene(s). In order to identify candidate TS gene for nasal NK/T cell lymphoma, we propose to execute the most extensive genetic study by using LOH and HOMOD analyses with 26 highly polymorphic microsatellite markers covering the entire chromosome 6q21-q25 region. LOH and HOMOD analyses have been performed on twelve paired and thirty-eight unmatched cases and several cases showed variable sizes of deleted fragments within this examined region. The smallest region of overlapping (SRO) deletion was determined and the potential TS region was narrowed down to an interval from D6S1633 to IGF2R that corresponds to 3.4 Mb. Roughly 68 known and hypothetical genes, as well as 50 Unigene clusters have been identified in this region and candidate TS genes among those will be studied further for their role in the pathogenesis of nasal NK/T cell lymphoma in the near future.
List of Tables……………………………………………………………I
List of Figures………………………………………………………….II
List of Abbreviation…………………………………………………..III
Abstract in Chinese……………………………………………………V
Abstract in English………………………………………………….VII


1. INTRODUCTION…………………………………………………….1
1.1 Cancer research in general……………………………………….1
1.2 Cytogenetic approaches in studying oncogenesis………………7
1.3 Molecular approaches in studying oncogenesis………………..12
1.3.1 Molecular cytogenetic approaches………………………….12
1.3.2 Molecular genetic approaches……………………………...14
1.4 The nasal NK/T cell lymphoma……………………………….17
1.4.1 Epidemiological studies…………………………………...17
1.4.2 Clinicopathological features……………………………….18
1.4.3 Association with EBV…………………………………….21

1.5 Previously identified genetic alterations of nasal NK/T cell lymphoma………………………………………………22
1.5.1 The chromosomal alterations identified by cytogenetic and molecular cytogenetic methods…………………………..22
1.5.2 The molecular genetic alterations identified by LOH analysis……………………………………………………...24
1.6 Purpose of this study………………………………………………27


2. MATERIALS AND METHODS……….………………..………..28
2.1 Samples collection………………………………...………………28
2.2 DNA extraction……………………………...…………………….28
2.3 Microsatellite markers selection and PCR condition……………31
2.4 Genotyping analysis and heterozygosity determination………...32
2.5 LOH analysis…………………………………………………….33
2.6 HOMOD analysis………………………………………………..35
2.7 Immunohistochemistry…………………………………………..37


3. RESULTS...……………………………………………………………39
3.1 Case summary……………………………………………………..39
3.2 Immunohistochemical staining of CD56……….…………..…….39
3.3 Heterozygosity determination of twenty-six selected microsatellite markers covering chromosome 6q21-25 in Taiwanese population....42
3.4 Multiplex genotype analysis………………………………………45
3.5 LOH analysis……………………………………….………….…..45
3.6 HOMOD analysis………………………………………….………47
3.7 Determination of smallest region of overlapping (SRO) deletions..48


4. DISCUSSION.…………………………………………………….…..53


5. REFERENCES…………….………………………………………….60
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