臺灣博碩士論文加值系統

EZH2是PcG蛋白群的一員，其功能在調節幹細胞的自我更新與分化。在PI(4,5)P2的合成過程PIP5K1C扮演著重要的角色。磷脂酶C（phospholipase C）會將PI(4,5)P2水解成細胞內傳訊者IP3和DAG，其中IP3是細胞內鈣離子的活化者。在神經分化過程中，細胞內鈣離子濃度改變是必須且關鍵的。然而，EZH2是否調控細胞內鈣離子訊息傳遞，以及它是如何調控人類間質幹細胞（hMSCs）的神經分化，其機制目前尚未清楚。在本研究中，偵測神經標記的表現結果顯示，我們成功地誘導hMSCs分化成神經系列（lineage）的細胞。將EZH2抑制後，細胞內鈣離子的濃度很顯著上升約14倍，也使PIP5K1C的表現量上升。在hMSCs細胞中，EZH2會跟PIP5K1C的啟動子（promoter）結合，而在神經分化後EZH2會離開。抑制PIP5K1C會降低EZH2默化細胞內鈣離子釋出，也會抑制hMSCs的神經分化，這顯示EZH2能藉由PIP5K1C調控細胞內鈣離子濃度，進而影響hMSCs的神經分化。至此，我們最早提出此證據顯示，在增生的hMSCs細胞中，EZH2負調控細胞內鈣離子。在神經分化的細胞中，EZH2離開PIP5K1C的啟動子後，活化PIP5K1C的表現，並誘導細胞內鈣離子訊息傳遞，使hMSCs分化成神經系列的細胞。

Enhancer of zeste homolog 2 (EZH2), a polycomb group (PcG) protein, regulates stem cells renewal and differentiation. Phosphatidylinositol-4-phosphate 5-kinase, type I gamma (PIP5K1C) plays an important role in synthesis of phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2]. Phospholipase C (PLC) hydrolyzes PI(4,5)P2 into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), in which IP3 is an intracellular messenger to activate calcium signaling. Change in intracellular calcium concentration is required and critical in neuronal differentiation. However, whether EZH2 modulates intracellular calcium signaling and how it regulates neuronal differentiation of human mesenchymal stem cells (hMSCs) are still unclear. In the present study, we successfully induce hMSCs to differentiate into neuronal lineage by detecting the expression of neuronal markers. Knockdown of EZH2 dramatically increases the level of intracellular calcium by 14-fold, and also evokes the expression of PIP5K1C. EZH2 binds the promoter of PIP5K1C in hMSCs and then dissociates after neuronal differentiation. Knockdown of PIP5K1C significantly reduces intracellular calcium release in EZH2-silenced cells, and disrupts neuronal differentiation of hMSCs, indicating that EZH2 regulates intracellular calcium through PIP5K1C, and thus affects neuronal differentiation of hMSCs. Here, we provide the first evidence to show in proliferating hMSCs, EZH2 negatively regulates intracellular calcium. After induction of neuronal differentiation, EZH2 departs from the promoter of PIP5K1C, activates its expression, and evokes intracellular calcium signaling, leading to differentiate hMSCs into neuronal lineages.

目錄…………………………………………………………………………………Ⅰ
圖目錄………………………………………………………………………………Ⅳ
表目錄………………………………………………………………………………Ⅵ
致謝…………………………………………………………………………………Ⅶ
縮寫表…………………………………………………………………………………1
中文摘要………………………………………………………………………………3
英文摘要………………………………………………………………………………4
第壹章 前言…………………………………………………………………………5
第一節 研究背景……………………………………………………………………5
一、幹細胞介紹
二、EZH2蛋白質
三、EZH2與細胞分化的關係
四、鈣離子與細胞分化
五、PIP5K基因與鈣離子
第二節 研究目的與假設…………………………………………………………10
第貳章 材料與方法………………………………………………………………11
第一節 實驗材料…………………………………………………………………11
一、細胞株
二、藥品試劑
三、儀器設備與器材
第二節 實驗方法…………………………………………………………………12
一、人類間質幹細胞（3A6）之培養及誘導其神經分化
二、細胞萃取物（lysate）的製備
三、蛋白質濃度之測定
四、西方墨點法
五、質體（plasmid）抽取
六、利用shRNA抑制基因表現
七、RNA之萃取
八、反轉錄作用（Reverse Transcription）
九、聚合酶鏈鎖反應(polymerase chain reaction, PCR)及DNA洋菜膠電泳分析
十、即時定量PCR（Real-time PCR）
十一、染色質免疫沉澱（chromatin immunoprecipitation）
十二、利用流式細胞儀（flow cytometry）分析細胞內鈣離子濃度
第参章 實驗結果……………………………………………………………………20
一、人類間質幹細胞的分化特性鑑定
二、慢病毒（Lentivirus）的製備與測試
三、EZH2在神經分化過程中的表現
四、EZH2蛋白調控hMSCs的PIP5K1C基因表現
五、在細胞分化不同時間點，其細胞內鈣離子濃度變化
六、EZH2調控細胞內鈣離子
七、EZH2透過PIP5K1C調控細胞內鈣離子的表現
八、PIP5K1C參與hMSCs的神經分化
九、PLC參與hMSCs的神經分化
第肆章 結論與討論…………………………………………………………………24
參考文獻……………………………………………………………………………27
圖表…………………………………………………………………………………33
附錄…………………………………………………………………………………54
圖 目 錄
圖一、顯微鏡觀察hMSCs的神經分化特性………………………………………33
圖二、以西方墨點法觀察hMSCs的神經分化相關蛋白表現……………………34
圖三、以RT-PCR觀察hMSCs的神經分化神經標記表現…………………………35
圖四、藉由GFP螢光反應觀察轉殖成效以及病毒表現情形………………………36
圖五、EZH2在神經分化過程中的表現……………………………………………37
圖六、利用ChIP方法證明EZH2蛋白結合至PIP5K1C的啟動子（promoter）…38
圖七、以RT-qPCR分析EZH2調控PIP5K1C的表現………………………………39
圖八、以西方墨點法分析EZH2調控PIP5K1C的表現……………………………40
圖九、在細胞分化不同時間點，細胞內鈣離子濃度變化…………………………41
圖十、抑制EZH2表現，對細胞內鈣離子濃度的影響……………………………42
圖十一、實驗模式……………………………………………………………………43
圖十二、抑制PIP5K1C表現，對細胞內鈣離子濃度的影響………………………44
圖十三、EZH2透過PIP5K1C調控細胞內鈣離子的表現…………………………45
圖十四、以RT-qPCR探討PIP5K1C基因於神經分化過程中的表現情形………46
圖十五、以西方墨點法探討PIP5K1C蛋白於神經分化過程中的表現情形………47
圖十六、藉由RT-qPCR分析shRNAs抑制PIP5K1C基因的表現………………48
圖十七、藉由西方墨點法分析shRNAs抑制PIP5K1C蛋白的表現………………49
圖十八、抑制PIP5K1C對神經分化的影響…………………………………………50
圖十九、抑制PLC對神經分化的影響………………………………………………51
圖二十、訊息傳導路徑………………………………………………………………52
圖二十一、HPCA與HPCAL1氨基酸序列比對……………………………………53
附圖一、間質幹細胞分化成不同細胞類型…………………………………………6
附圖二、Polycomb蛋白複合體的調控使基因的表現受抑制………………………7
附圖三、三種不同型態的PIP5K1……………………………………………………8
附圖四、PIP5K是PI(4,5)P2的主要合成酶…………………………………………9
附圖五、IP3誘導細胞內鈣離子由ER釋出至細胞內………………………………9
附圖六、研究假設……………………………………………………………………10
表 目 錄
附表一、RIPA溶液的組成…………………………………………………………54
附表二、SDS-PAGE的配製…………………………………………………………55
附表三、PCR使用的primer…………………………………………………………56
附表四、RT-qPCR使用的primer……………………………………………………57
附表五、初級抗體（primary antibody）……………………………………………58
附表六、二級抗體（secondary antibody）…………………………………………59

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