Polycystin-2為細胞膜上可通透鈣離子之陽離子通道，由pkd2基因轉譯而成。在臨床醫學上發現當pkd2 基因發生突變會導致人類先天性遺傳疾病多囊性腎病 (autosomal dominate polycystic kidney disease, ADPKD)，造成患者腎臟產生囊腫，嚴重者會導致腎衰竭進而死亡。本研究是利用斑馬魚 (Danio rerio) 作為實驗動物，研究pkd2 基因缺損對斑馬魚胚胎腎臟與心臟發育之影響。以半定量反轉錄聚合?連鎖反應 (semi-quantitative RT-PCR) 分析斑馬魚pkd2 基因在不同胚胎發育時期之表現情形，發現pkd2 基因在斑馬魚胚胎在1-細胞期已有表現，而在8hpf 至12hpf 表現量上升，其餘各時期表現量皆維持穩定直到48hpf。以RNA全覆式原位雜交 (RNA whole-mount in situ hybridization) 偵測斑馬魚pkd2 基因在斑馬魚胚胎12hpf 時開始廣泛表現於胚胎的全身，在16hpf 與30hpf 可觀察到pkd2 基因在胚胎頭部及軀幹均有表現，且多集中表現於頭部。40hpf、48hpf 及72hpf 可觀察到 pkd2 基因除了在頭部及軀幹有表現外在胚胎腎管也可偵測到pkd2 mRNA。利用專一性反意股寡核酸抑制pkd2基因的表現後，由組織切片可觀察到胚胎出現腎管囊腫。胚胎外觀上則出現包括腦水腫、心包膜水腫、腹部水腫以及軀幹彎曲等現象。進一步可觀察到胚胎心臟產生缺損且胚胎存活率降低。利用半定量RT-PCR 偵測pkd2 基因弱化胚胎的心臟發育相關標誌基因cmlc2、amhc、vmhc 及tbx5 mRNA 表現情形，結果顯示在24hpf、30hpf 及48hpf 胚胎之cmlc2、amhc、vmhc 及tbx5 mRNA 表現量均有顯著下降，顯示pkd2 基因弱化直接或間接影響心臟生成與發育。在腎臟功能測試實驗中，於斑馬魚胚胎48hpf、72hpf、96hpf 及120hpf 注射10 kD dextran-rhodamine 觀察到pkd2 基因弱化後72hpf、96hpf 及120hpf 胚胎體內螢光量有累積的現象。在改變體外滲透壓實驗中，將pkd2 基因弱化之胚胎在48hpf 由胚胎培養液中改浸泡入125 mOsm/L 或350 mOsm/L 濃度蔗糖溶液中，24 小時後發現浸泡125 mOsm/L 蔗糖溶液之胚胎水腫情形並無明顯改變。浸泡350 mOsm/L 蔗糖溶液之胚胎水腫現象則有輕微減少，但無法完全消除。以原子吸光儀測定胚胎體內離子含量實驗中，偵測到pkd2 基因弱化之胚胎魚體內鈉離子含量有顯著上升之現象。本研究結果亦證明pkd2 基因弱化後可導致斑馬魚胚胎腎臟衰竭，進而引發心臟異常及心腎綜合症 (cardiorenal symdrom)。而本研究結果亦顯示pkd2 基因弱化後所造成斑馬魚胚胎全身性不可逆性胚胎水腫現象可能是經由四種因素相包括：心臟發育缺損、腎臟功能受損、胚胎滲透壓失調及胚胎體內鈉離子失衡互作用所生成的結果。以斑馬魚DNA 晶片偵測pkd2 基因弱化後斑馬魚胚胎在48hpf 基因表現情形，本研究總共篩選43663 個基因，其中14309 個基因為有效基因，在有效基因中共挑選出了217 個基因表現量上升兩倍以上，112 個基因表現量被抑制兩倍以下。透過此一結果可了解pkd2 基因弱化後其他基因表現可能調控機制與作用路徑，做為日後找尋腎病生成原因之依據。本研究證明以斑馬魚建立pkd2 基因弱化及慢性腎臟衰竭模式的可行性。將來可應用於pkd2 基因與腎臟疾病之相關研究。

謝辭 i
中文摘要............................................... iii
英文摘要............................................... v
目錄................................................... vii
表目錄................................................. xii
圖目錄................................................. xiii
壹、前言................................................ 1
一、腎臟 (Kidney) .................................. 1
二、PKD2 基因及其所屬家族......................... 8
三、腎臟與心臟疾病發生之關連性..................... 12
四、心臟 (Heart).................................... 13
五、水腫 (edema) 形成與各種生理缺損之關係........... 18
六、應用DNA 微陣列晶片 (DNA microarray) 在斑馬魚胚胎發育上之研究................................ 21
七、本研究之目標................................... 23
貳、實驗材料............................................ 26
?、實驗方法............................................ 29
一、斑馬魚飼養與受精胚胎之收集及培育............... 29
1、斑馬魚之飼養............................... 29
2、斑馬魚受精卵之收集及培育................... 29
二、斑馬魚pkd2基因之選殖........................... 29
1、萃取斑馬魚胚胎之全量核糖核酸 (Total RNA) ... 29
2、核糖核酸電泳分析........................... 30
3、引子 (Primer) 設計.......................... 31
4、反轉錄?反應 (Reverse Transcription) .......... 31
5、聚合?連鎖反應 (Polymerase Chain Reaction, PCR) ................................... 32
6、膠體萃取 (Gel Extraction) .................... 32
7、勝任細胞 (Competent Cell) 製備............... 33
8、接合作用 (Ligation).......................... 34
9、質體轉型作用 (Transformation) ................ 34
10、篩選 (Selection) ............................ 34
11、小量質體製備 (Mini-preparation).............. 35
12、分析與定序 (Auto-sequence) ................. 35
三、斑馬魚pkd2 RNA 在不同發育時期之定量分析....... 36
1、萃取斑馬魚胚胎之全量核糖核酸 (Total RNA) ... 36
2、半定量反轉錄?反應 (Semi-quantitative RT-PCR). 37
四、RNA全覆式原位雜交 (RNA Whole-mount in situ Hybridization)偵測斑馬魚pkd2 基因之表現位置...... 37
1、斑馬魚胚胎之收集、固定與保存............... 37
2、核醣核酸探針 (RNA probe) 之製備............ 38
3、抗DIG抗體之預吸附 (Preabsorption) 反應...... 40
4、RNA全覆式原位雜交 (RNA Whole-mount in situ Hybridization) ............................. 41
五、顯微注射 (Microinjection)......................... 44
1、訂購顯微注射用pkd2 專一性反意股寡核酸 (Morpholino Antisense Oligos, MO) ........... 44
2、顯微注射 (Microinjection) pkd2 專一性反意股寡核酸...................................... 44
六、利用pkd2 專一性反意股寡核酸抑制綠螢光蛋白表現............................................ 45
1、pkd2-GFP 載體構築.......................... 45
2、顯微注射 (Microinjection) pkd2 專一性反意股寡核酸與 pkd2-GFP 載體...................... 47
七、利用pkd2 專一性反意股寡核酸弱化pkd2 基因外顯子 (Exon) 表現................................... 47
1、顯微注射pkd2 專一性反意股寡核酸........... 47
2、檢測pkd2 專一性反意股寡核酸弱化pkd2 基因外顯子表現胚胎與正常胚胎mRNA 表現差異..... 48
八、胚胎發育觀察與結果之計算方式................... 48
九、組織切片觀察弱化pkd2 基因表現後對腎臟結構之影響............................................ 49
1、 斑馬魚胚胎之收集、固定與保存.............. 49
2、胚胎脫水與浸潤............................. 50
3、胚胎包埋 (Embedding) ....................... 50
4、切片 (Section) .............................. 50
5、切片染色................................... 51
十、以心臟發育相關之標誌基因，研究弱化pkd2 基因表現後對心臟發育之影響............................ 51
1、萃取斑馬魚胚胎之全量核糖核酸 (Total RNA) ... 51
2、半定量反轉錄聚合?連鎖反應 (Semi-quantitative RT-PCR) .................................. 51
十一、注射10-kD rhodamine-dextrane 觀察弱化pkd2 基因表現後對腎臟功能之影響....................... 52
1、訂購注射用10-kD rhodamine-dextrane........... 52
2、顯微注射10-kD rhodamine-dextrane............. 52
十二、利用浸泡蔗糖溶液觀察改變環境滲透壓對胚胎水腫發生之影響................................... 52
十三、利用原子吸光儀 (Atomic Absorption) 偵測pkd2 基因弱化後胚胎體內鈉離子變化情形............... 53
十四、運用斑馬魚晶片 (Danio rerio DNA Microarray) 探討pkd2 基因弱化後心臟相關基因的表現情形........ 53
1、萃取斑馬魚胚胎之全量核糖核酸............... 53
2、反轉錄?反應 (Reverse Transcription) .......... 53
3、轉錄作用及標定螢光染劑 (Transcription and Coupling with Fluorescent Dye) ................ 53
4、純化cRNA (Purifying cRNA) .................. 54
5、雜交 (Hybridization) 前處理.................. 54
6、雜交反應 (Hybridization) ..................... 54
7、晶片洗滌 (Microarray Wash) .................. 54
8、晶片掃描 (Scanning) ......................... 55
9、晶片結果分析 (data analysis) .................. 55
肆、結果................................................ 56
一、斑馬魚pkd2 基因在不同胚胎發育時期之表現模式.... 56
二、RNA 全覆式原位雜交 (RNA whole-mount in situ hybridization) 偵測斑馬魚pkd2 基因在不同胚胎發育時期之表現位置................................... 56
三、利用pkd2 專一性反意股寡核酸 (Morpholino antisense oligos，MO) 顯微注射 (microinjection) 抑制pkd2 蛋白表現及利用pkd2 專一性反意股寡核酸弱化pkd2 基因外顯子 (exon) 表現.............................. 57
四、利用pkd2-MO 弱化pkd2 基因表現後，觀察斑馬魚胚胎不同發育時期之活存率......................... 57
五、組織切片觀察弱化pkd2 基因表現後對腎臟結構之影響............................................. 58

六、利用pkd2 專一性寡核酸弱化pkd2 基因表現後，觀察斑馬魚胚胎缺損情形之比較....................... 58
七、注射不同pkd2 專一性反意股寡核酸弱化pkd2 基因表現後，計算斑馬魚胚胎不同發育時期之存活率及外觀缺損之比較....................................... 60
八、以心臟發育相關之標誌基因，研究弱化pkd2 基因表現後對心臟發育之影響............................. 61
九、注射10-kD rhodamine-dextrane 觀察弱化pkd2 基因表現後對腎臟功能之影響.......................... 61
十、利用浸泡蔗糖溶液觀察改變環境滲透壓對胚胎水腫發生之影響................................... 62
十一、利用原子吸光儀 (Atomic Absorption) 偵測pkd2 基因弱化後胚胎體內鈉離子變化情形............... 63
十二、運用斑馬魚晶片 (Danio rerio DNA microarray) 探討pkd2 基因弱化後心臟相關基因的表現情形........ 63
伍、討論................................................ 65
一、斑馬魚pkd2 基因在不同胚胎發育時期之表現模式.... 65
二、RNA 全覆式原位雜交 (RNA whole-mount in situ hybridization) 斑馬魚pkd2 基因在不同胚胎發育時期之表現位置....................................... 65
三、利用pkd2 專一性反意股寡核酸 (Morpholino antisense oligos, MO) 顯微注射 (microinjection) 抑制pkd2 蛋白表現........................................... 66
四、利用pkd2-MO 弱化pkd2 基因表現後，觀察斑馬魚胚胎不同發育時期之活存率......................... 67
五、組織切片觀察弱化pkd2 基因表現後對腎臟發育之影響............................................. 67
六、利用pkd2 專一性寡核酸弱化pkd2 基因表現後，觀察斑馬魚胚胎發育異常情形之比較................... 68
七、設計並注射不同pkd2 專一性反意股寡核酸弱化pkd2 基因表現後，計算斑馬魚胚胎不同發育時期之存活率及外觀缺損之比較................................. 70
八、以心臟發育相關之標誌基因，研究弱化pkd2 基因表現後對心臟發育之影響............................. 70
九、注射10-kD rhodamine-dextrane 觀察弱化pkd2 基因表現後對腎臟功能之影............................. 71
十、利用浸泡蔗糖溶液觀察改變環境滲透壓對胚胎水腫發生之影響............................. 73
十一、pkd2 基因弱化對斑馬魚胚胎體內離子平衡 (ionic balance) 之影響............................... 74
十二、運用斑馬魚晶片 (Danio rerio DNA microarray) 探討pkd2 基因弱化後心臟相關基因的表現情形........ 74
陸、參考文獻............................................ 79
圖表................................................... 91
附錄................................................... 124

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