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博碩士論文 etd-0215112-152652 詳細資訊
Title page for etd-0215112-152652
論文名稱
Title
雄性激素受體聚集體在胚胎幹細胞分化期間的作用
The roles of androgen receptor aggregates in embryonic stem cell differentiation
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
82
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2012-01-11
繳交日期
Date of Submission
2012-02-15
關鍵字
Keywords
雄性激素受體、雄性激素受體聚集體、胚胎幹細胞、伴護蛋白、內質網壓力
embryonic stem cell, AR, chaperones, ER stress, AR aggregates
統計
Statistics
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The thesis/dissertation has been browsed 5718 times, has been downloaded 1197 times.
中文摘要
雄性激素受體(AR)為固醇受體家族成員之一,而雄性激素受體基因內CAG(編碼形成胺基酸glutamine,簡稱Q)重複序列的延伸是引起一些神經肌肉退化性疾病的主因,如Spinal bulbar muscular atrophy (SBMA) 脊髓延髓肌肉萎縮症或Kennedy disease,SBMA疾病的特徵是細胞核附近能形成poly-glutamine AR(poly-Q AR)的蛋白質聚集體,稱為“雄性激素受體聚集體”(AR aggregates)。先前的研究顯示,在過量表現正常型態AR的轉殖鼠中,它的骨骼肌肉纖維卻能被觀察出有和SBMA疾病的樣本相似的不正常表現型態。為了更了解正常型態蛋白形成的聚集體造成對細胞具毒性的機制,本實驗使用沒有基因修飾的正常老鼠胚胎幹細胞於雄性激素受體過量表現情況下呈現poly-Q疾病的細胞常見特徵,此實驗中發現,胚胎幹細胞在雄性激素反應之下,AR蛋白質被高度表現並有核內聚集體inclusion bodys形成的現象,而雄性激素受體聚集體的形成抑制類囊胚體的分化,並在雄性激素引起的細胞凋亡中提升caspase-3蛋白的活性。
此外,本實驗也觀察在胚胎幹細胞中伴護蛋白、雄性激素受體和內質網壓力活化途徑之間的關係,而發現伴護蛋白能與AR aggregates位置重合,這些發現也許對了解伴護蛋白作用於AR aggregates上所扮演的角色有很大的幫助。
Abstract
Androgen receptor (AR) is a member of the steroid hormone receptor family of molecules, and expansion of a CAG repeat encoding polyglutamine (poly-Q) in AR gene are associated with a progressive neuromuscular disease known as spinal bulbar muscular atrophy (SBMA) or Kennedy disease. The hallmark of SBMA diseases is formation of juxtanuclear AR inclusions that have been termed ‘AR aggregates’.Previous studies showed that transgenic mice overexpressing wild-type AR exclusively in the skeletal muscle fibers display similar abnormalities to those observed in models of SBMA disease.
To elucidate the mechanisms underlying toxicity conferred by wild-type protein aggregation within normal cells, a mouse embryonic stem cell (ESC) model with non-genetic modified settings in AR overexpression was used to display the common features of polyglutamine disease in this experiment. It was found that wild-type AR proteins are highly expressed and form nuclear aggregate inclusions in response to androgen treatment in ES cells, the formation of AR aggregates inhibit the differentiation of embryonic bodys and enhanced caspase-3 activity in androgens -induced apoptosis.
In addition, it was also investigated that relation between chaperones、AR and the endoplasmic reticulum (ER) stress-induced pathways in ES cells in this study, and it was found that chaperones could colocalize with AR aggregates, these findings may help us to better understand the roles of the chaperones on AR aggregates.
目次 Table of Contents
論文審定書…………………………………………………i
中文摘要……………………………………………………ii
英文摘要……………………………………………………iii
第一章、前言………………………………………………1
第一節、胚胎幹細胞………………………………………1
第二節、雄性激素與雄性激素受體………………………2
第三節、蛋白質的摺疊……………………………………3
第四節、雄性激素受體聚集體和神經退化性疾病………4
第五節、內質網壓力與不完全摺疊蛋白反應……………6
第六節、伴護蛋白…………………………………………7
第二章、材料與方法……………………………………..10
第一節、實驗設計………………………………………..10
第二節、試驗試劑………………………………………..10
第三節、小鼠胚胎幹細胞的培養………………………..10
第四節、轉染……………………………………………..11
第五節、charcoal-treated serum 的製作……………..12
第六節、caspase-3 assay.…………………………….12
第七節、XBP-1 splicing RT-PCR assay……………...13
第八節、免疫沉澱法………………………………..……13
第九節、西方墨點分析………………..…………………14
第十節、免疫螢光染色…………………………………..16
第十一節、Filter trap assay.................…………………17
第十二節、Real-time PCR..……………………………18
第十三節、小鼠胚胎幹細胞類囊胚體的培養………..…18
第十四節、實驗統計分析…………………………..……19
第三章、結果與結論……………………………..………20
第一節、小鼠胚胎幹細胞分化過程中,DHT提升AR表現量並伴隨著AR aggregates的增加……………………...20
第二節、過量的雄性激素受體誘導AR aggregates表現增加…………………………………………………………...20
第三節、高濃度DHT抑制小鼠類囊胚體分化成骨骼肌細胞…………………………………………………………..21
第四節、雄性激素DHT增加caspase-3所誘導的細胞凋亡…………………………………………………………..22
第五節、DHT/AR能活化不摺疊蛋白反應………………23
第六節、DHT提升伴護蛋白GRP78和AR結合…………24
第四章、討論…………………………………………… 26
第一節、在小鼠胚胎幹細胞中,雄性激素/雄性激素受體所調節的細胞凋亡...............................................................26
第二節、雄性激素受體對小鼠胚胎幹細胞分化的影響及生理意義……………………………………………………...27
第三節、伴護蛋白 GRP78 對雄性激素受體聚集體的作用…………………………………………………………...29
參考資料…………………………………………………...31
附錄…………………………………………………… …..60
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