非轉譯CAG重複序列擴增之轉殖基因鼠在肌肉與睪丸中其基因及蛋白質表現之差異性分析

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题名:	非轉譯CAG重複序列擴增之轉殖基因鼠在肌肉與睪丸中其基因及蛋白質表現之差異性分析 Identification of the differentially expressed genes and proteins in the muscle and testis of transgenic mice containing untranslated CAG trinucleotide repeats expansion
作者:	張佑誠 Iou-Cheng Chang
贡献者:	中山醫學大學：醫學研究所潘惠錦
关键词:	三核酸重複序擴增突變非轉譯區蛋白質二維膠體電泳 trinucleotide repeat disorders untranslated regions
日期:	2005/07/07
上传时间:	2009-12-01T02:41:32Z (UTC)
摘要:	三核酸重複序擴增突變(expansion mutation)會導致人在經及肌肉退化方面的遺傳疾病，主要造成影響的序為CAG及與之互補的CTG。CAG擴增突變通常是發生在密碼區(coding region)；CTG擴增突變通常是發生在非轉譯區(untranslated region；UTR)。實驗室在之前即開始以同長的CAG重複序為對象，將重複序接在螢光蛋白(EGFP)基因之3’端轉譯區(3’untranslated region)，並以gamma-sarcoglycan(GSG)基因的promoter使EGFP在小鼠肌肉專一性表達。初步結果已顯示3’UTR的(CAG)n三核酸重複序的擴增突變在(CAG)200的確會造成鼠肌肉病片組織型態常、骨骼肌發生孿縮、活動較差、蟲線體結構改變，生育能低等情形。本研究中我們分成轉及轉譯的個層面探討CAG重複序擴增存在對於小鼠中肌肉與睪丸組織所造成的影響。在轉方面，以cDNA subtraction與cDNA microarray種技術分析基因受調控的情形。經由RT-PCR及Real-Time PCR鑑定結果，我們找到受到up-regulated基因有5個，down-regulated基因有2個，這些基因大多與細胞分化、能代謝有關。在轉譯方面，以蛋白質二維膠體電泳技術分析蛋白質受調控的情形，以MALDI-TOF-MS質譜儀鑑定受影響蛋白質種，找到在肌肉組織中受到up-regulated蛋白有10個，down-regulated蛋白有4個，這些蛋白質同樣大部分與細胞分化以及能代謝有關。另外，在睪丸組織中找到up-regulated蛋白有1個，down-regulated蛋白有7個，這些蛋白質主要是和成作用有關。這是首次以基因體與蛋白質體整合的概，探討3’ UTR 的CAG重複序擴增時，在肌肉與睪丸中基因及蛋白質受到調控的現象。由我們的結果顯示，CAG重複序擴增對於細胞的影響在轉譯方面要比轉方面的明顯，這樣的結果或許可以為後續對於非轉譯重複序擴增疾病的研究上提供一個新的方向。 Among human trinucleotide repeat disorders, the most frequent triplets found to be expanded are CAG and its complementary sequence, CTG. CAG repeats are almost always found on coding regions whereas CTG expansions are located in untranslated regions (UTRs). Previously we made transgenic mice expressing a muscle-specific transcript with (CAG)200 inserted in the 3’-UTR of EGFP gene. Mice express expanded CAG repeat exhibited abnormal muscle cell morphology, low muscle activity and decreased reproduction ability. In this study, we investigated the effects of CAGn expansion on the transcription and translation of muscle and testis in mice. In the aspects of transcription, we used techniques of cDNA subtraction and cDNA microarray to analyze how genes are regulated.Results from RT-PCR and Real-Time PCR revealed five up-regulated genes, and two down-regulated genes. In the aspects of translation, we compared the differential expressed proteins by 2D-gel electrophoresis and identified proteins by MALDI-TOF-MS. Using this approach, ten up-regulated proteins and ten down-regulated proteins were found. Most of these genes and proteins are related to differentiation and metabolism. Furthermore, we also found one up-regulated and seven down-regulated proteins in testis, and these proteins are mainly related to spermatogenesis. This is our first study to integrate the genomic and proteomic concepts to investigate the regulation of genes and proteins in muscle and testis of transgenic mice with CAG repeats expansion in UTR. Our data indicate that the effects of CAG repeat expansion is more obvious on translation than transcription. This tendency may offer a new direction for the research of trinucleotide repeat expansion disease in the future.
URI:	http://140.128.138.153:8080/handle/310902500/287
显示于类别:	[醫學研究所] 博碩士論文

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