先前CGG重複擴增所引起的Fragil X syndrome的細胞模式研究，
顯示未轉譯的CGG重複擴增對於人類細胞是具有毒性的，並改變
caspase-8、CYFIP、Neurotensin、UBE3A等基因的表現(Handa et al., 2005)。故本研究利用MG-132和staurosporine兩種藥物來處理淋巴細胞 株，藉此探討正常人與帶有SCA8 CTG重複擴增的病人淋巴細胞對藥
物的敏感差異如何。MG-132可抑制蛋白質降解的ubiquitin-proteasome 的路徑(Myung et al., 2001)，而ubiquitin-proteasome pathway在細胞的 訊息傳遞、週期調節、轉錄或是DNA修復等的功能上扮演重要的角色 (Ciechanover, 1994; Glickman and Ciechanover, 2002)。Staurosporine為 一種protein kinase C的抑制劑，可使細胞透過caspase走向細胞凋亡的 路徑。實驗結果發現三位患者細胞株在經過proteasome抑制劑MG-132 或細胞自戕刺激物staurosporine處理後，在MG-132 200 nM及STS 50 nM的濃度下，與未處理相較，細胞死亡率顯著上升，而年齡、性別 配對的正常人細胞株則無此現象(圖十)。顯示帶有SCA8 CTG重複擴
增的病人淋巴細胞對此兩種藥物的敏感度較高。因此，更進一步利用 staurosporine 50 nM濃度處理淋巴細胞，來探討caspase-3活性之差異。
Caspase 3為一種aspartate-specific cysteine proteases，在功能方面為 xecutioner caspase，須透過次單位的剪裁活化，使細胞走向凋亡路徑 (Fuentes-Prior and Salvesen, 2004)。首先利用西方轉漬法來探討藥物處
理過後的正常人與病人淋巴細胞，其活化的caspase-3蛋白的差異。可 能由於caspase-3蛋白的低表現量及/或表現差異不夠大，導致西方轉漬 法無法判別差異(data not shown)。進一步利用高敏感度的EnzChek Caspase-3 Assay Kit來偵測caspase-3活性，結果發現病人淋巴細胞株在 staurosporine 50 nM濃度處理下，其caspase-3活性有顯著增高，而正常 人則無明顯差別(圖十一)。結果顯示病人淋巴細胞株在此藥物濃度處 理下，可能會透過caspase-3走向細胞凋亡的路徑。故本研究顯示SCA8 CTG重複擴增對於人類細胞是具有毒性的，其所影響的基因，值得深 入探討問題。
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陸、附錄圖表
圖一、ATXN8OS、ATXN8與KLHL1的基因構造圖與CpG島預測分 析。(A) ATXN8OS
、
ATXN8與KLHL1基因構造圖。ATXN8OS與KLHL1 兩個基因的啟動子、表現子1及部分intron 1重疊，導致mRNA 5'端分 別表現自同一段DNA的兩股，且ATXN8OS mRNA有選擇性裁接的現 象，CTG重複位於可以被選擇性裁接的表現子A上。此外，ATXN8與 KLHL1的轉錄為同一方向，CAG重複位於表現子A上，可轉錄出polyQ蛋白 (圖形修改自Mutsuddi and Rebay, 2005)。(B) ATXN8OS與ATXN8 啟動子及5'端區域的CpG島預測分析。彎曲箭頭+1標示處為ATXN8OS 轉錄起始位置，標示ATG處為ATXN8 polyQ蛋白轉譯的起始點。藍色 長方形標示處為CpG島。
圖二、SCA8 CTG 三核苷酸重複等位基因頻率(A)及多型性基因型(B) 分佈圖。所檢測的台灣人族群包括正常人(Controls)、帕金森氏症患者 (Parkinson’s disease)、原發性顫抖症(Essential tremor)及其他神經相關 疾病患者與其家屬(Others)。
圖三、PCR-based限制酶檢測之引子設計。ATXN8OS之genomic DNA (exon D ~ intron D) (A)和cDNA (exon D ~ C1) (B)序列，可能的CpG雙 核苷(cg)以粉紅色標示出，進行PCR-based限制酶檢測的ATXN8OS基 因引子(F2、R2)和ATXN8OS cDNA基因引子(F2、R3)以底線標示出，
切割酵素HpaII、MspI之切割位(ccgg) 以紅色長方形標示。
圖四、H327 和 H600 家族之 ATXN8OS 基因甲基化分析。(A)基因組 DNA 未添加酵素(-)、添加甲基化敏感酵素 HpaII (H)、添加甲基化不 敏感酵素 MspI (M)處理的 0.8%瓊脂膠體電泳分析照片。(B)以上述處 理後DNA 為模板之 PCR 產物(ATXN8OS 基因 5'UTR 區域，464 bp) 的1.4%瓊脂膠體電泳分析圖，PCR 增幅 SPINK5 基因 exon 29 343 bp 片段作為內在對照組。(C) H327 和 H600 家族的甲基化程度量化圖。
cDNA 基因 內生性基因
圖五、SCA8 (CR)0~157胚胎腎臟細胞株的甲基化分析。(A)基因組DNA 未添加酵素(-)、添加甲基化敏感酵素HpaII (H)、添加甲基化不敏感
酵素MspI (M)處理的0.8%瓊脂膠體電泳分析照片。(B)以上述處理後 DNA為模板之PCR產物(ATXN8OS cDNA基因5'端，494 bp；ATXN8OS 內生性基因5'UTR區域，464 bp)的1.4%瓊脂膠體電泳分析圖，PCR增 幅SPINK5基因exon 29 343 bp片段作為內在對照組。(C) cDNA基因(白 色長柱)和內生性基因(黑色長柱)的甲基化程度量化圖。
圖六、SCA8 (CR)23~157胚胎腎臟細胞株的Bisulfite定序分析。可能的 CpG位置以黑色底線標示出。PCR-based限制酶檢測所辨認的三個 HpaII、 MspI酵素切割位置(C↓CGG)以藍色長方形標示出。
圖七、免疫染色質沈澱-PCR之引子設計。ATXN8OS之cDNA (exon B ~ A) (A)和genomic DNA (exon A ~ 3’ flanking) (B)序列，進行免疫染色 質沈澱-PCR的ATXN8OS cDNA基因引子(F4、R4)和ATXN8OS基因引 子(F5、R5)以底線標示出。
圖八、SCA8 (CR)23~157胚胎腎臟細胞株的 CHIP-PCR 分析。(A)基因 組 DNA 經超音波震盪前(-)、後(+)的 0.8%瓊脂膠體電泳圖。以抗體 anti-dimethyl H3-K9 (B)或 anti-acetyl H3-K14 (C)進行 CHIP-PCR 後產 物的1.4%瓊脂膠體電泳圖，lane P 未經 CHIP 步驟，lane -未加抗體、
但經 CHIP 步驟，lane +加抗體、且經 CHIP 步驟，PCR 產物大小為 213-bp (exon B ~ exon A)或 494-bp (exon D ~ exon C1) ATXN8OS cDNA 片段。