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研究生: 侯懿婷
Yi-Ting Hou
論文名稱: 退化性神經疾病:PPP2R2B基因族群遺傳分析及分生研究
Neurodegenerative disease:PPP2R2B gene genetics study and molecular research
指導教授: 李桂楨
Lee, Guey-Jen
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2005
畢業學年度: 93
語文別: 中文
論文頁數: 56
中文關鍵詞: 脊髓小腦運動失調症第12型PPP2R2B
英文關鍵詞: SCA12
論文種類: 學術論文
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  • 脊髓小腦運動失調症(Spinocerebellar ataxias, SCAs)是一群體染色體顯性遺傳的神經退化性疾病,小腦功能異常是所有類型的共同症狀,且其中許多類型亦會引起中樞或是周邊神經系統的損傷。第十二型脊髓小腦運動失調症(SCA12)為PPP2R2B基因5'端CAG三核重複擴增的疾病。PPP2R2B基因會轉譯出一個專一表現在腦部的去磷酸PP2A (protein phosphotase 2A)的調控次單位Bβ,來調節PP2A對vimentin、histone-1、tau等蛋白的去磷酸化活性。本研究的目的在探討PPP2R2B基因CAG三核重複的遺傳及功能性角色。本研究分析了阿茲海默氏症、帕金森式氏症、運動失調症、精神病患者及正常人族群的PPP2R2B基因CAG三核重複的分佈頻率,發現CAG三核酸重複範圍沒有顯著差別,但阿茲海默氏症樣品群的(CAG)7的比例顯著高於其他樣品群。人類腦癌細胞株SK-N-SH、LA-N-1、IMR-32、胚胎腎細胞HEK-293及大鼠ST14A細胞的啟動子功能性分析顯示,CAG三核上游序列而非下游序列的缺失,會減低其啟動子活性。CAG三核重複的擴增(49及64個三核)會使PPP2R2B基因啟動子活性增加二至三倍,但罕見的(CAG)7 對偶基因表現的活性,僅為族群中常見的10、13、16個三核對偶基因的60%。CAG重複的變異可能影響鄰近啟動子element的相對距離,進而影響其啟動子活性。PPP2R2B基因啟動子(CAG)7 對偶基因可能為一功能性的多型性,影響個體對阿茲海默氏症的感受性。

    The hereditary spinocerebellar ataxias (SCAs) are a group of autosomal dominant adult onset neurodegenerative disorders. Cerebellar dysfunction is a hallmark of all the SCAs, and most also involve other regions of the central or peripheral nervous system. SCA12 is caused by a CAG repeat expansion in the 5' UTR of the PPP2R2B gene. The PPP2R2B gene encodes a brain-specific regulatory subunit Bβ of PP2A (protein phosphotase 2A). Bβ regulates PP2A dephosphorylation activity for specific substrates, including vimentin, histone-1, and tau. The purpose of this study was to determine the genetic and functional roles of the CAG repeat polymorphism within the PPP2R2B gene. The CAG repeat was analyzed in patients with Alzheimer's disease, Parkinson's disease, ataxia, schizophrenia, and healthy controls. The distribution of the alleles in patients was not significantly different from that in the controls, whereas the (CAG)7 allele was significantly overrepresented in the AD patients compared to the controls. The promoter functional assay showed that the deletion of the upstream sequence, but not the downstream sequence of the CAG repeat, reduced the PPP2R2B promoter activity. Expansions of the repeat (49 and 64 triplets) caused a 2~3-fold increase in PPP2R2B promoter activity and the rare (CAG)7 allele displayed 60% activity, as compared to the common 10-, 13-, and 16-triplet alleles, respectively, in human SK-N-SH, LA-N-1, IMR-32, HEK-293 and rat ST14A cells. The upstream sequence of the CAG repeat, but not the CAG repeat or its downstream sequence, was essential for the PPP2R2B transcription. Variations in the CAG repeat number may alter the spacing of flanking promoter elements to influence the expression of PPP2R2B. The (CAG)7 may be a functional polymorphism in the 5' promoter region of PPP2R2B and may be associated with the susceptibility to AD.

    目錄……………………………………………………………………….I 中文摘要……………………………………………………..................IV 英文摘要……………………………………………………..................VI 圖次表………………………………………………………………...VIII 壹、緒論   一、脊髓小腦運動失調症………………………………..................1   二、脊髓小腦運動失調症第十二型……………………..................3   三、PPP2R2B基因的族群遺傳分析………………………….…….5   四、PPP2R2B的功能…………..…………………………………....6   五、PPP2R2B基因啟動子…………………………………………..7   六、PPP2R2B的可能致病機轉……………………………………..9   七、研究動機……………………………………………………... 10 貳、研究材料與方法   一、研究樣品…………………………………………………..…..11   二、基因組DNA (genomic DNA)的萃取…………………………11   三、聚合連鎖反應(PCR) ……………………………………….12   四、PPP2R2B基因5'端CAG三酸重複的基因型分析      (genotyping)…………………………………………………..12 五、PPP2R2B基因啟動子片段的選殖及定序          (一)多型性啟動子片段的製備……………………..………..13     (二)自洋菜膠中純化DNA片段………………………….….13     (三)接合反應………………..……………………………......14     (四)轉形勝任細胞之製備……..………………………...…...14     (五)細菌的轉形作用……………………………….………...15     (六)質體DNA的小量置備及DNA定序…………..……..…16     (七) DNA定序…………………………………………...…..16   六、PPP2R2B基因啟動子片段CAG重複的擴增………..………17   七、pGL3-TK/PPP2R2B-CAG26重組質體的構築………………..17   八、缺失不同部位的啟動子質體的構築…………………………18   九、質體DNA的大量置備及純化…………………….…………..19   十、細胞培養………………………………………………………19   十一、PPP2R2B基因啟動子的功能性分析………………………20   十二、RNA的萃取………………………………………………...21   十三、Reverse transcription PCR………………………….........….21   十四、同步定量PCR (Real Time PCR)…………………………...22 參、結果   一、PPP2R2B基因CAG三核酸重複遺傳資料庫的建立……..24   二、細胞株中內生性PPP2R2B的表現量………………………...25 三、轉移細胞株中PPP2R2B的相對表現量……………………...25   四、CAG三核酸重複變異對啟動子活性的影響……………...26 五、CAG三核重複鄰近序列對PPP2R2B基因的轉錄調節…..27 肆、討論   一、台灣地區PPP2R2B基因CAG三核酸重複的遺傳資料庫..29 二、各細胞株中內生性及轉移PPP2R2B基因的表現…………...32   三、CAG三核重複鄰近序列對於PPP2R2B基因的轉錄調節..33   四、CAG三核重複變異對PPP2R2B基因啟動子活性的影響..35 五、PPP2R2B基因低啟動子活性與阿茲海默氏症相關性………36 六、結論……………………………………………………………36 伍、參考文獻……………………………………………………………38 陸、附錄圖表……………………………………………………………44

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