臺灣博碩士論文加值系統

人類脊髓肌肉萎縮症 (SMA) 是一種運動神經元退化的疾病，目前並沒有有效的治療方式。為了進一步了解此疾病的致病機轉，本實驗室首先利用基因轉殖與剔除的技術建立了SMA-like小鼠動物模式。透過利用SMA-like小鼠的胚胎期纖維母細胞(MEF)以及人類SMN2基因轉染的小鼠運動神經元細胞株(SMN2-NSC34)的體外篩選，我們發現sodium vanadate、TSA、aclarubicin三種藥能夠透過誘導Stat5蛋白質來增加SMN2基因的表現。在人類SMN2-NSC34細胞中，Stat5蛋白質的表現能夠增加SMN2啟動子的活性，使得SMN2基因同時產生較多的全長型式以及缺少7號外顯子 (exon) 的轉錄產物。此外減少Stat5蛋白質的表現會干擾SMN2的活化但無法影響藥物所造成的SMN2基因剪接型式的改變，顯示Stat5的作用是在於SMN2基因的轉錄調控上。在EB病毒轉形的第一型SMA病人的淋巴細胞中，表現持續活化的Stat5蛋白質能夠改善病人nuclear Gems減少的現象。另一方面，Stat5也能夠改善SMA小鼠胚胎期運動神經元細胞神經軸突伸展的缺陷。這些結果證明Stat5的訊號傳遞路徑可以作為將來在SMA治療上的藥物設計標的。
此外，我們也發現了sodium vanadate在SMN2-NSC34細胞株中會誘導Sfrs9基因差異性的表現，轉錄出只含有RNA結合片段的SRp30c蛋白質 (RRM-SRp30c)。RRM-SRp30c蛋白質雖然缺少一般SR蛋白質所具有的RS功能性區域，然而經由免疫螢光染色分析，我們發現其在細胞內表現的型式與其他SR蛋白質相似，顯示其仍具有SR的性質。進一步將RRM-SRp30c蛋白質與SMN2 mini-gene載體共同表現在NSC34中，或是將RRM-SRp30c蛋白質表現在有SMN2-NSC34細胞株中，均可以發現其能影響SMN2基因的選擇性剪接作用。我們進一步發現RRM-SRp30c蛋白質能夠與作用在SMN2基因7號外顯子上的Tra2β1蛋白質以及辨識在3’-剪接位置(3’-splice site)與內含子(intron)上的連續嘧啶區域 (polypyrimidine tract) 的U2AF35/65蛋白質交互作用。這個結果也顯示RRM-SRp30c蛋白質可能是與這些剪接作用因子共同形成一個複合體，調控SMN2基因的剪接反應過程，幫助其7號外顯子的編入。

SMA is a motor neuron degeneration disorder for which there is currently no effective treatment. In this study, we have identified three compounds, sodium vanadate, TSA, and aclarubicin that effectively enhance SMN2 expression through inducing Stat5 activation in SMA-like mouse embryonic fibroblasts and human SMN2 transfected NSC34 cells. First, we found that Stat5 activation enhanced SMN2 promoter activity with increase of both full-length and exon7-deleted SMN transcripts in SMN2-NSC34 cells. Knockdown of Stat5 expression disrupted the effects of sodium vanadate on SMN2 activation but did not influence SMN2 splicing, suggesting that Stat5 signaling is involved in SMN2 transcriptional regulation. In addition, constitutive activation of Stat5 mutant (Stat5A1*6) profoundly increased the number of nuclear gems in SMA-patient lymphocytes and reduced SMA-like motor neuron axon outgrowth defects. These results demonstrate that Stat5 signaling could be a possible pharmacological target for SMA treatment designing.
We also identified an alternative form of SRp30c which was induced by sodium vanadate in NSC34 cells and contributed to SMN2 exon7 inclusion. Sodium vanadate induced an alternative expression form of Sfrs9 which containing a novel included exon. The large Sfrs9 transcript however may translate a RS domain truncated SRp30c which remain retained its RNA recognition motif (RRM-SRp30c) and expressed high level in brain but not in other tissues, suggesting a specific regulation for Sfrs9. Furthermore, the RRM-SRp30c partially associates with SRp30c speckle, indicates its potential role involved in mRNA splicing. The RRM-SRp30c over-expression enhanced exon7 inclusion in SMN2 minigene transfected NSC34 cell or stably expressed of human SMN2 NSC34 cell and it also associates with Tra2β1 and the U2AF35/65. This demonstrates its involvement in the formation of a splicing complex which mediates SMN2 exon7 inclusion.

縮寫表……………………………………………………………………8
附圖目錄………………………………………………………………..10
第一章 Stat5持續活化表現能改善人類脊髓肌肉萎縮症的缺陷…..12
第一節………………………………………………………………..12
壹、 人類脊髓肌肉萎縮症(SMA)概述…………………………..12
貳、 SMN 基因與調控…………………………………………...13
參、 SMN蛋白質…………………………………………………14
一. SMN蛋白質的特性………………………………………14
二. SMN蛋白質的house keeping功能………………………15
三. SMN蛋白質的其他可能功能……………………………17
四. SMN蛋白質在神經細胞中扮演的角色…………………17
肆、 SMA的治療概況 …………………………………………..19
一、 SMA的小鼠動物治療模式……………………………...19
二、 HDAC 抑制劑 ………………………………………….19
三、 其他的藥物與治療方式 ………………………………...21
第二節、材料與方法………………………………………………..22
第三節、結果…………………………………………………………33
壹、建立藥物篩選的體外(in vitro)分析系統…………………….34
貳、三種有效藥物影響Stat家族蛋白的表現……………………34
參、Stat5調控SMN2基因的表現………………………………..36
肆、抑制Stat5表現並不能影響SMN2基因的選擇性剪接作用..38
伍、 Stat5持續活化能改善SMA的缺陷………………………..39
第四節、討論…………………………………………………………42
第二章 缺少RS-domain的SRp30c蛋白質受到sodium vanadate誘導表現並刺激SMN2基因第7號外顯子的編入……………45
第一節、 前言………………………………………………………..45
壹、 選擇性剪接作用與SR蛋白質……………………………..45
貳、 SMN基因的選擇性剪接作用………………………………49
第二節、 實驗材料與方法…………………………………………..51
第三節、 結果………………………………………………………..57
壹、 Sodium vanadate誘導Sfrs9基因在NSC34細胞中差異性的表現………………………………………………………….57
貳、RRM-SRp30c蛋白質仍具有SR蛋白質特性並參與SMN2基因的剪接作用……………………………………………….59

參、 RRM-SRp30c增加SMN2基因7號外顯子的編入……….60
肆、 RRM-SRp30c蛋白質與Tra2-β1、U2AF蛋白質作用…….62
第四節、 討論………………………………………………………..63
圖表……………………………………………………………………..66
參考資料………………………………………………………………105
附錄……………………………………………………………………127

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