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研究生:傅胤傑
研究生(外文):Yin-Chieh Fu
論文名稱:建立雄性素受體於神經系統活化基因表現之生物模式
論文名稱(外文):Modeling of Androgen Receptor Activated Gene Transcription in Nervous System
指導教授:黃銓珍
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:英文
論文頁數:46
中文關鍵詞:雄性素受體前列腺特異抗原神經系統
外文關鍵詞:androgen receptorPSASK-N-BE(2)nervous system
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雄性素除了對於男性泌尿生殖系統的生長發育、性行為上具有重要功能外,亦能影響其他組織的發育及生長,如:肌肉、神經、骨骼…等等。睪固酮(testosterone)及dihydrotesterone為兩種已知於人體中具生物活性之雄性素。雄性素與其受體(AR)結合後構型改變,AR與原先相結合的蛋白質脫離,改而結合另一群蛋白質形成新的蛋白質複合體。活化的AR進入細胞核,辨識DNA上特定序列稱為androgen response element (ARE),協同其他輔助因子調控基因表現。序列分析結果發現前列腺特異抗原(PSA)啟動子區域存在數個ARE,其基因表現受雄性素所活化。因此,利用PSA來研究AR調控基因表現機制是很廣泛被使用的模式。
將PSA啟動子上游加強子移除後,近端1.5 kb PSA啟動子能組織特異性地表現在斑馬魚的神經、骨骼、及肌肉系統。同時,近端1.5 kb PSA啟動子在神經系統衍生而來的細胞株,可經由雄性素刺激而有較強表現能力。基於上述實驗結果,我們利用近端1.5 kb PSA啟動子啟動報導基因表現,作為研究AR於神經系統調控基因表現的工具。一方面利用綠色螢光蛋白作為報導基因,建立in vivo 斑馬魚模式,研究環境荷爾蒙對神經系統發育的影響;另一方面利用螢火蟲螢光酵素(luciferase)作為報導基因,以SK-N-BE(2)細胞建立體外細胞模式,期望藉由報導基因表現的變化,來研究AR在神經系統上調控基因表現及神經分化過程的功能與機制。
首先將人類AR表現載體轉染到SK-N-BE(2)細胞,得到AR持續表現細胞株(SK-AR clones)。其中SK-AR62細胞表現高量AR、呈現雄性素依賴生長,但一般常用來抑制雄性素功能的抗雄性素卻無法拮抗此一雄性素促進細胞生長的的功能。進一步將近端1.5 kb PSA啟動子啟動報導基因表現載體轉染入SK-AR62細胞,或將AR與報導基因同時表現的載體轉染至SK-N-BE(2)細胞得到的報導細胞株:SK-AR-Luc及SK-PLAR 細胞株。然而,有報導基因表現的細胞株,其報導基因表現不受雄性素影響,呈現持續性表現。雖然在報導基因細胞株建立上並未得預期結果,但我們得到SK-AR62及SK-PLAR54兩株細胞,分別可藉由retinoic acid或血清去除的處理方式,誘導突觸伸長、進行神經分化,使細胞在型態上更趨近於神經細胞,提供作為研究AR在神經分化過程中所扮演的功能很好的材料。
Androgens are steroids critical for the development and maintenance of the male phenotype. Androgens are known to regulate functions of various tissues, including muscular tissue, nervous system, bone, and sexual behavior. Testosterone (T) and its metabolite dihydrotestosterone (DHT) are well-known androgens exert their effects through binding to the androgen receptor (AR), which is a transcription factor. Hormone binding promotes the nuclear translocation of the AR, where AR recognizes certain DNA sequence called androgen response element (ARE) and regulates gene expression. AR positively regulates the transcription of prostate specific antigen (PSA) mainly through binding to AREs in the proximal region and the PSA distal enhancer region of PSA promoter. To understand androgen functions in the nervous system, we introduced GFP, reporter gene, regulated by PSA promoter into zebrafish embryos. Examine GFP expression in zebrafish embryos by using fluorescent microscope, revealing that PSA1.5 promoter expresses significantly in notochord, muscle, and neuronal tissues. Androgens not only play an important role in reproductive tissues, but also in nervous system during neurogenesis. Indeed, neuronal expression of PSA promoter is further confirmed by using transient transfection in different human neuronal cell lines. We want to set up a model to study AR mediated genomic effect in nervous cells by using PSA1.5 promoter as a tool. Here we choose human neuroblastoma cell line SK-N-BE(2) as an in vitro model because it may differentiate into neuron-like phenotype and has been studied in estrogen receptor regulated neuron differentiation. Human AR are introduced into SK-N-BE(2) cells by stable transfection derived SK-AR62 line. Further characterization showed that SK-AR62 grows in an androgen-dependent manner. However, anti-androgen cannot inhibit DHT induced cell proliferation in SK-AR62. Stable transfection of PSA1.5-Luc into SK-AR62 results in constitutive expression of luciferase disregard of hormone treatment. Furthermore, introduction of plasmids harboring AR expression cassette and PSA1.5-Luc reporter into SK-N-BE(2) to derive SK-PLAR clones, results in constitutive expression of luciferase as aforementioned result. Among the SK-PLAR clones studied, SK-PLAR54 can differentiate neuron-like morphology upon 3 days culture in serum free medium. Overall, these results indicate PSA1.5-Luc expresses androgen-independently in the presence of AR in SK-N-BE(2) derived cells. The AR may activate the 1.5Kb PSA promoter ligand-independently in SK-N-BE(2) cells.
Contents
Contents I
Figures III
Abbreviation IV

中文摘要 1
Abstract 3
Introduction 4
Androgens and androgen receptor 4
Prostate-specific antigen (PSA) 7
Zebrafish as an animal model 8
Neuroblastoma 10
Specific aims 11
Materials and methods 12
Materials 12
Methods 12
PCR amplification 12
Plasmid constructs 12
Plasmid DNA preparation 13
Zebrafish 13
Microinjection of zebrafish embryos 13
Fluorescene microscopic observation 14
Cell culture 14
Cell subculture 15
Reporter gene transfection and luciferase assay 15
Stable transfection of cells 15
Protein extraction and quantification 16
Sodium dodecyl sulfate polyacrylamide gel electrophoreis
(SDS-PAGE) 16
Western blotting 17
Immunofluorescene cell staining 17
Crystal violet staining 18
Results 19
Expression pattern of a GFP gene driven by the PSA promoter in zebrafish embryos 19
Zebrafisfh harboring 1.5kb-PSA-GFP transgene stable
line establishment 17
PSA promoter transcriptional activity in prostate and
breast cancer cell lines 20
PSA promoter transcriptional activity in neuronal
cancer cell lines 20
SK-AR stable line establishment 21
Androgen-dependent cell proliferation of SK-AR62 21
Introduction of PSA1.5-Luc reporter gene into SK-AR62 22
SK-PLAR stable line establishment 22
Serum free treatment induced SK-PLAR54 cells
differentiation 23
Retinoic acid treatment induced SK-AR62 cells
differentiation 23
Discussion 25
Reference 41
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