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

張佑誠(Iou-Cheng Chang)

指導教授 ： 潘惠錦

中山醫學大學/醫學院/醫學研究所/碩士(2004年)

摘要

三聯核酸重複序列擴增突變(expansion mutation)會導致人類在神經及肌肉退化方面的遺傳疾病，主要造成影響的序列為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不同長度的CAG重複序列為對象，將重複序列接在綠螢光蛋白(EGFP)基因之3’端不轉譯區(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三聯核酸重複序列的擴增突變在(CAG)200的確會造成老鼠肌肉病理切片組織型態異常、骨骼肌發生孿縮、活動力較差、精蟲粒線體結構改變，生育能力降低等情形。 本研究中我們分成轉錄及轉譯的兩個層面來探討CAG重複序列擴增存在對於小鼠中肌肉與睪丸組織所造成的影響。在轉錄方面，以cDNA subtraction與cDNA microarray兩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、能量代謝有關。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類，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量代謝有關。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精作用有關。 這是首次以基因體與蛋白質體整合的概念，探討3’ UTR 的CAG重複序列擴增時，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重複序列擴增對於細胞的影響在轉譯方面要比轉錄方面來的明顯，這樣的結果或許可以為後續對於非轉譯重複序列擴增疾病的研究上提供一個新的方向。

關鍵字

三聯核酸重複序列擴增突變 ； 非轉譯區 ； 蛋白質二維膠體電泳

並列摘要

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.

並列關鍵字

trinucleotide repeat disorders ； untranslated regions

參考文獻

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