臺灣博碩士論文加值系統

糖尿病是一種不能夠有效利用葡萄糖當作能量來源的疾病。一般而言，糖尿病主要有兩種類型:第一型(胰島素依賴型)糖尿病與第二型(非胰島素依賴型)糖尿病。第一型糖尿病主要是因病人的免疫系統異常，攻擊分泌胰島素的貝它細胞，使得體內貝它細胞(βcells)破壞殆盡，因此無法分泌或分泌不足胰島素以調控血糖濃度。這一類型的病人需要每日注射胰島素才能生存。最近的臨床實驗顯示，胰島移植可以成為另一種更有效率而且具有永久性的治療第一型糖尿病的方法。然而缺乏可供移植的胰島使得這項治療方法的推廣變得非常緩慢。因此，如果我們能瞭解胰臟幹細胞在胚胎發育中生長與發育的機制，我們便能發展出培養胰臟幹細胞和誘導其分化的方法，進而利用這個方法大量生產胰島，以供人體移植之用。在以前的實驗中，本實驗室辨認出一個在胚胎胰臟中有獨特表現型式的新基因，命名它為“內胚層衍生激素”(Endoderm Derived Factor，EDF)。從這個基因轉譯出的蛋白質一級序列來判斷，可能是一種被分泌至細胞外的激素，其大小約為55 KDa。在過去研究指出，EDF在老鼠胚胎9.5、10.5天表現於胃及胰臟。然而，胚胎12.5天卻只表現在胃部而胰臟已不再表現。我的碩士論文研究主題在於EDF對於胰臟發育上所扮演的生物角色為何，以下是我研究EDF的方法:
1. 再次確認EDF在胚胎各不同胚胎時期所表現的位置。2. 製備EDF多株抗體，
以供往後實驗所需。3. 為了日後觀測EDF活性，因此先在293T細胞表現。4. 製作EDF基因轉植老鼠，觀察胰臟大量表現EDF後對其發育的影響。到目前為止，我的實驗結果支持過去研究所指出EDF在各不同胚胎時期表現的位置。同時也證實了EDF確實是一種分泌型蛋白分子。再者，EDF多株抗體及轉植老鼠也已經完成。

Diabetes is a disorder of metabolism-the way our bodies use digested food for growth and energy. Type 1 diabetes is an autoimmune disease in which the body’s immune system mistakenly attacks its own insulin-producing beta cells and eventually kills them. The pancreas then produces little or no insulin. A person who has type 1 diabetes must take insulin daily to live. Scientists have made many advances in islet transplantation for treating type1 diabetic patients in recent years, but a major obstacle to widespread use of islet transplantation will be the shortage of islet cells. Therefore, if we can understand the mechanisms that regulate the growth and differentiation of pancreatic stem cells, it would be feasible to grow these cells in vitro and induce them to differentiate into islet cells, and thus provide sufficient pancreatic islets for transplantation.From a previous differential screening experiment, we have identified a novel gene expressed in the early developing pancreas in a specific pattern, named Endoderm Derived Factor (EDF). This gene encodes a protein of 55 KDa with a secretion signal peptide sequence at its N-terminus, suggesting that it may function as a secreted signaling molecule. From preliminary data, EDF appears to be expressed in pancreas and stomach at E9.5 or E10.5, but only expressed in stomach at E12.5. My master thesis project had investigated the biological role of EDF in pancreas development, which includes: 1. reconfirming the expression pattern of EDF in embryos of different developing stages. 2. generating anti-EDF antibody for future application. 3. testing the activity of EDF in 293T cell. 4. generating a transgenic mouse line that over-express EDF and observe its effects on pancreas development.Thus far, immunostain and in situ hybridization of EDF has been performed and the detected EDF expression pattern is consistent with previous results. I have generated anti-EDF antibody and the transgene construct, and were demonstrated that EDF is indeed a secreted molecule.

中文摘要…………………………………………………………………… 3
英文摘要…………………………………………………………………… 4
目錄………………………………………………………………………… 5
圖目錄……………………………………………………………………… 53
表目錄……………………………………………………………………… 75
附錄……………………………………………………………………… 71
第一章 前言
第一節 胰臟的生理簡介……………………………………… 7
第二節 胰臟疾病的介紹……………………………………………… 8
第三節 糖尿病的簡介………………………………………………… 9
第四節 糖尿病的治療………………………………………………… 10
第五節 胰臟的發育…………………………………………………… 11
第六節 轉錄因子(transcription factors)和胰臟的發育………… 12
第七節 Morphogen signaling in early pancreas development…………… 15
第八節 Notch signaling pathway in pancreas development……………… 16
第九節 Sonic hedgehog signaling pathway in pancreas development…… 17
第十節 研究動機…………………………………………………… 18
第二章 材料與方法
第一節 質體的純化…………………………………………………… 20
第二節 胚胎組織切片的製備………………………………………… 21
第三節 胚胎組織切片………………………………………………… 21
第四節 Invitro-transcription of EDF…………………………………… 22
第五節 原位雜合……………………………………………… 22
第六節 免疫染色法…………………………………………………… 25
第七節 質體的構築…………………………………………………… 26
第八節 GST融合蛋白質的表現……………………………………… 30
第九節 GST融合蛋白質的純化……………………………………… 31
第十節 從膠體純化EDF-GST融合蛋白…………………………… 31
第十一節 蛋白質定量………………………………………………… 32
第十二節 免疫轉印法………………………………………………… 32
第十三節 細胞培養…………………………………………………… 33
第十四節 細胞之質體轉染…………………………………………… 33
第十五節 免疫沉澱………………………………………………… 34
第十六節 alkaline phosphatase酵素活性分析………………………… 34
第十七節 從細胞培養液小量純化EDF蛋白質……………………… 35
第十八節 stable clone 製備…………………………………………… 35
第十九節 成鼠器官genomic DNA製備……………………………… 36
第二十節 EDF基因轉殖老鼠引子專一性實驗……………………… 36
第二十一節 篩選EDF基因轉殖老鼠………………………………… 37
第三章 結果
第一節 EDF抗體置備………………………………………………… 38
第二節 EDF抗體測試………………………………………………… 39
第三節 細胞內表現EDF……………………………………………… 39
第四節 原位雜合與免疫染色法觀察老鼠胚胎EDF的表現………… 40
第五節 EDF基因轉植老鼠的確認…………………………………… 41
第四章 討論……………………………………………………………… 43
參考文獻…………………………………………………………………… 47
結果圖片…………………………………………………………………… 53

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