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標題: | 正常成人周邊血液單核球細胞之染色體重組 Chromosomal Rearrangements Detected in Peripheral Blood Mononuclear Cells of Normal Adults |
作者: | Jian-Ching Tu 涂建勍 |
指導教授: | 張淑媛(Sui-Yuan Chang) |
共同指導教授: | 常蘭陽 |
關鍵字: | 正常成人,周邊血液,染色體重組,轉位, normal adult,PBMC,chromosomal rearrangement,translocation, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 人類基因體的變異主要分為單一核苷酸多型性 (SNPs) 位點以及結構變異(Structural Variation) ;後者包含了刪除 (deletion) 、插入 (insertion) 、倒置 (inversion) 、轉位 (translocation) 與重複 (duplication) 。至今已發現約一千萬個SNPs位點變異,來研究人類遺傳以及表型的變化。此外,人類基因體變異數據資料庫 (Database of Genomic Variants, DGV) 將較大片段的結構變異資料建置資料庫,到2010年趨近九萬筆資料,以拷貝數變異佔最多,約99%。目前對結構變異偵測的主要技術為array-CGH,其可偵測拷貝數改變的結構變異,但卻無法偵測平衡結構變異。平衡結構變異包含倒置及轉位。實驗室先前所建立的技術Restriction Enhanced Capturing Of Rearranged DNA (RECORD) 以inverse PCR為基礎,配合限制酵素切除未轉位模板。在目標基因上做結構變異的偵測,配合序列分析,來決定結構變異。先前已利用RECORD技術在人類精蟲細胞中偵測三種基因之轉位,對在新生兒及幼兒型血癌常見的轉位基因MLL,及幼兒型骨癌常見的轉位基因EWSR1,和兒童型淋巴性白血病轉位基因TCF3。許多研究發現MLL染色體轉位斷裂點呈明顯叢聚分佈;此外,根據之前研究,NHEJ-deficient B細胞在進入周邊組織後,抗體再修飾的發生也會造成染色體的重組,觀察到MYC基因與免疫球蛋白的轉位。本論文的主要研究方向為,以人類的周邊血液單核球細胞Peripheral Blood Mononuclear Cells (PBMCs) 作為實驗材料。利用RECORD技術配合特異性引子,偵測MLL基因在正常人的體細胞是否與生殖細胞具有相同染色體重組之現象。為了確認實驗條件,我們發現RECORD技術可偵測到在RS4;11細胞株帶有t(4;11)(q21;q23)的平衡轉位染色體,帶有相互的轉位的第四對及第十一對染色體。之後,利用RECORD分析25位沒有血癌的正常成人所抽出的周邊血液,來檢測正常人體細胞中是否具有不正常的轉位染色體的存在。實驗結果發現,在不同的正常人的PBMCs細胞中,可以偵測到隨機轉位的染色體結構。其轉位的對象隨機散佈在不同染色體的位置上,由序列方向性推測可以分為三種類型之染色體,為雙中心 (dicentric)、無中心 (ancentric)、單中心 (derivative) 的染色體。細胞複製時的檢查點會使非單中心染色體之細胞無法複製,影響體細胞所偵測之染色體轉位以單中心佔最多。接著由前述25位正常人中隨機挑選10位,利用CD3/CD19螢光抗體標定T及B細胞,經由流式細胞儀分選系統將單核細胞分為B、T、non-B/non-T三類細胞後,同樣利用RECORD偵測轉位,觀察轉位的發生是否在不同細胞間存在有差異。由本研究中發現,生殖細胞的染色體重組的機制,可以保留到個體發育後的細胞內。此機制提供演化的推力而保留於個體,但是否某些偶發性的基因相關疾病與此機制具有相關性,則需要更深入的探討。 Variations in the human genome consists of single nucleotide polymorphisms (SNPs) sites, and structural variation (SVs); which contains deletion, insertion, inversion, translocation and duplication. Up to date, more than 10 million SNPs have been used to study human genetic and phenotypic changes. In addition, data on human genome variation database (Database of Genomic Variants, DGV) to larger fragments of the structural variation information data has collected 90,000 document information, among which 99% is copy number variation. The current technology array-CGH can detect changes of copy number variation, but not the balanced structural variation, including inversion and translocation. Our laboratory has previously established the technique Restriction Enhanced Capturing Of Rearranged DNA (RECORD) to determine the target genes structure variation and was shown to detect three kinds of gene translocation, the neonatal and infant leukemia common translocation gene MLL, child care type of bone cancer common translocation gene EWSR1, and child-type lymphoblastic Leukemia translocation gene TCF3 in human sperm cells. In this study, we set to determine whether chromosomal rearrangement also occur in human Peripheral Blood Mononuclear Cells (PBMCs) using MLL as target gene. The RS4; 11 cell lines with t(4; 11)(q21; q23) balanced translocation of chromosomes was first used to determine the experimental conditions and sensitivity of the RECORD. Among the PBMCs from 25 healthy individuals, some random translocation chromosome structure was observed. The object of its translocation chromosomes was scattered in different locations, and the orientation suggested by the sequence can be grouped into three types of chromosomes, the two-center (dicentric), no center (acentric), and single-center (derivative) chromosomes. Flow cytometry sorting was next used to determine the cell types, B, T, and non-B/non-T groups, in which translocations occur. Our preliminary results suggest that germ cell chromosomal rearrangement mechanisms can be retained to differential cells. Our study results provide some information about the mechanism of evolution within individual, yet whether it can be related to some sporadic gene-related diseases and the potential mechanisms require further exploration. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22328 |
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顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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