臺灣博碩士論文加值系統

在人體之中，胰臟負責維持內分泌系統、外分泌系統以及代謝的恆定。之前研究指出，要發展成為一個可以正常運作的胰臟必頇由許多轉錄因子的調控，其中包含PDX1、ptf1a和Ngn3等，而這些重要的轉錄因子經由許多研究的探討後，可以結合成一個聯絡網。其中在胰臟內分泌細胞發育的過程中，Ngn3扮演很重要的角色，已經有研究指出當缺乏Ngn3的情形之下無法成功產生內分泌系統的前驅細胞。
然而對於Ngn3調控胰臟內分泌系統的分子機制還不清楚。因此先前實驗室利用酵母菌雙雜交系統 (Yeast two-hybrid assay) 來尋找與Ngn3有交互作用的蛋白，經過篩選之後我們找到幾個基因，其中包含了CCAR1，接著我們就想來探討CCAR1對於Ngn3所扮演的角色。由於之前實驗室已經證實Ngn3與CCAR1具有交互作用，但是由酵母菌雙雜交系統篩選到的CCAR1只有胺基酸538～723的片段，並非全長。不過在這186個胺基酸之中仍然包含兩個不同的domain，分別是SAP domain和EDK-rich domain。所以我更進一步找出Ngn3所結合的CCAR1片段，經由實驗後發現在CCAR1中的EDK-rich domain是Ngn3結合的部位。接著關於CCAR1的功能分析，我使用實驗室之前建立的報告基因檢測 (report assay) 來分析。從螢光素酶報告基因檢測 (Luciferase reporter assay) 的結果顯示，當CCAR1的表現量遭到抑制而降低時，Ngn3的活化能力也隨著下降，進一步造成Luciferase活性跟著下降。另一部份若是將CCAR1的表現量增加則Ngn3的活化能力也跟著增加，相對影響Luciferase活性上升。這些結果顯是在Ngn3調控內分泌細胞分化的過程之中，CCAR1扮演調節的功能。
另一部分，我們推測Neurogenin family其餘兩個成員 (Ngn1和Ngn2) 能與CCAR1進行交互作用，並且我們也利用pull-down assay證實這項假設。而pull-down assay的結果只能代表從細菌所純化出來的Ngn1和Ngn2融和蛋白對於CCAR1具有直接的交互作用，但是為了更證實此交互作用形成的可能性，我們未來必頇利用動物細胞株進行共同免疫沉澱 (co-immunoprecipitation) ，才能更確保此交互作用的可能性。並且之後也可更進一步來探討CCAR1對於神經發育過程的影響。

In our body, pancreas plays a critical role in maintaining endocrine, exocrine, and metabolism homeostasis. Recent studies have identified a network of transcription factors, including Pdx1, Ptf1a, and Ngn3, regulating the development of pancreas. Particularly, Neurogenin-3 (Ngn3) has been shown to be required for the development of all endocrine cell lineages of the pancreas. Besides, Ngn3-deficient mice fail to generate any endocrine progenitor cells.
However the molecular mechanisms controlling the specification of pancreatic endocrine precursors remained unknown. Previously, our laboratory has utilized the yeast two-hybrid assay to find the proteins which can interact with Ngn3. After screening, we have identified several genes, including CCAR1, which can interact with Ngn3. Hence, we want to explore the role of CCAR1 for Ngn3. Because of the interaction between Ngn3 and CCAR1 has confirmed. But the CCAR1 from screening of yeast two-hybrid assay is amino acid 538~723, △CCAR1, not the CCAR1 full lentgh. Although the △CCAR1 had 186 amino acid but it included two different domain, SAP-domain and EDK-rich domain. Then, in my study, I want to prove which domain Ngn3 bound to. I have confirmed the protein interactions between CCAR1-EDK-rich domain and Ngn3 using pull-down assay. And about the functional assay of CCAR1, I used report assay to find the role of CCAR1 in pancreatic development. From our data of Luciferase reporter assay, the effect of Ngn3 would be down-regulated when the expression of CCAR1 was knocked down. And in the other hand, when we increased the expression of CCAR1, the activity of Luciferase would be raised by Ngn3 was up-regulated. Hence, it is also likely that CCAR1 may mediate pancreatic endocrine differentiation together with Ngn3.
Besides, we speculated that CCAR1 is direct targets of Ngn1 and Ngn2. We confirmed the hypothesis by pull-down assay. But the result of pull-down assay just explained the direct interaction between the purified protein of Ngn1, Ngn2, and CCAR1. In the future, we should use co-immunoprecipitation assay to prove the interaction. And then, we could also found the effect of CCAR1 in neuron development.

目錄
中文摘要
Abstract
第一章、序論 (INTRODUCTION AND BACKGORUND)
1-1 胰臟的結構與功能：
1-1-1 外分泌系統 (Exocrine System)：
1-1-2 內分泌系統 (Endocrine System)：
1-1-3 胰管 (Pancreatic Ducts)：
1-2 胰臟的發育：
1-3 胰臟發育時期中一些重要的轉錄因子：
1-3-1 PDX1的介紹：
1-3-2 Ptf1a (p48)的介紹：
1-3-3 Ngn3的介紹：
1-4 CCAR1的介紹：
1-5 神經的結構與功能：
1-5-1 中樞神經系統 (central nervous system, CNS)：
1-5-2 周邊神經系統 (peripheral nervous system, PNS)：
1-6 神經的發育：
1-7 神經元素家族 (Neurogenin family)：
1-8 研究動機：
第二章、材料與方法(MATERIALS AND METHODS)
2-1 進行質體構築前的DNA製備
2-1-1 RNA的萃取：
2-1-2 反轉錄作用 (Reverse transcriptase polymerase chain reaction；RT-PCR)：
2-1-3 聚合酵素鏈鎖反應 (Rolymerase chain reaction；PCR)：
2-1-4 限制酶反應 (Restriction enzyme digestion)：
2-1-5 接合反應 (Ligation)：
2-2 在細菌 (Bacteria) 表現Ngn1，Ngn2，Ngn3和CCAR1質體的構築：
2-3 在哺乳細胞 (Mammalian cell) 表現Ngn1， Ngn2， Ngn3和CCAR1質體的構築：
2-4 誘導目標蛋白質在大腸桿菌的表現
2-4-1 小量測試蛋白質的表現：
2-4-2 大量誘導蛋白質表現和萃取蛋白質：
2-5 GST和HIS融合蛋白的透析與BSA蛋白質的粗定量：
2-6 進行SDS-PAGE gel分析：
2-6-2 SDS-PAGE的染色及褪染：
2-6-3 SDS-PAGE的膠體乾燥保存：
2-7 GST-fusion protein pull down assay：
2-8 西方墨點分析法 (Western-blotting analysis)：
2-9 細胞的操作：
2-9-1 本研究所使用的細胞株：
2-9-2 解凍細胞：
2-9-3 更換培養液：
2-9-4 細胞分盤 (subculture)：
2-9-5 凍細胞：
2-10 細胞株的質體短暫植入 (Transient transfection)：
2-11 細胞株的蛋白質萃取：
2-12 蛋白質定量：
2-13 病毒的製備：
2-14 病毒的感染 (Infection) ：
2-15 螢光素酶報告基因檢測 (Luciferase reporter assay) ：
第三章、實驗結果(RESULT)
3-1 利用細菌系統表現Ngn3及CCAR1融合蛋白：
3-2 Ngn3能與CCAR1-E/D/K-rich domain有直接的交互作用：
3-3 利用CCAR1融合蛋白製備CCAR1多株抗體：
3-4 CCAR1抗體測試：
3-5 CCAR1能影響Ngn3調控NeuroD轉錄的能力：
3-6 利用細菌系統表現Ngn1及Ngn2融合蛋白：
3-7 Ngn1及Ngn2能與CCAR1有直接的交互作用：
第四章、討論(DISCUSSION)
第五章、參考資料(REFERENCES)
第六章、圖 (Figure)：
圖一A、細菌內表現Ngn3-FL質體示意圖。
圖一B、細菌內表現CCAR1-FL質體示意圖。
圖一C、細菌內表現△CCAR1質體示意圖。
圖一D、細菌內表現CCAR1-SAP domain質體示意圖。
圖一E、細菌內表現CCAR1-E/D/K-rich domain質體示意圖。
1. Ngn3-FL-His Tag：(amino acid：2-214)
2. GST-CCAR1-FL：(amino acid：1-1146)
3. GST-△CCAR1：(amino acid：538-723)
4. GST-CCAR1-SAP domain：(amino acid：630-670)
5. GST-CCAR1-E/D/K-rich domain：(amino acid：667-897)
圖二、由細菌所產生Ngn3-His Tag與不同長度的GST-CCAR1融合蛋白示意圖。
圖三A、Ngn3-FL-His Tag融合蛋白的表達及純化。
圖三B、GST-CCAR1 -FL融合蛋白的表達及純化。
圖三C、GST-△CCAR1融合蛋白的表達及純化。
圖三D、GST-CCAR1-SAP domain融合蛋白的表達及純化。
圖三E、GST-CCAR1-E/D/K-rich domain融合蛋白的表達及純化。
圖四、GST-△CCAR1 融合蛋白濃縮後與不同濃度BSA 定量。
圖五、利用HEK 293T細胞株產生的內生性蛋白進行CCAR1多株抗體測試。
圖六、內生性CCAR1的表現量。
圖七、Ngn3和CCAR1-E/D/K-rich domain有直接交互作用。
圖八A、細胞內表現Ngn3-FL質體示意圖。
圖八B、細胞內表現CCAR1-FL質體示意圖。
圖八C、細胞內表現NeuroD promoter 質體示意圖。
圖九、降低CCAR1的表現會影響Ngn3調控NeuroD promoter轉錄的能力。
圖十、增加CCAR1的表現量能影響Ngn3調控NeuroD promoter轉錄的能力。
圖十一A、細菌內表現Ngn1-FL質體示意圖。
圖十一B、細菌內表現Ngn2-FL質體示意圖。
圖十二A、Ngn1-FL-His Tag融合蛋白的表達及純化。
圖十二B、Ngn2-FL-His Tag融合蛋白的表達及純化。
圖十三、Ngn1、Ngn2與CCAR1有直接交互作用。
圖十四、Ngn1和CCAR1-E/D/K-rich domain有直接交互作用。

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