臺灣博碩士論文加值系統

在胰臟的藍氏小島(胰島)中有一群被命名為β-cells的細胞，這種具有分泌胰島素能力的細胞佔整個胰島的細胞總數高達65~80%，胰島素是調控血糖的重要內分泌賀爾蒙，而第一型糖尿病目前被認為可能是由於β-cells的缺失或是失去弁鄔瓴伬P的，誘導β-cells再生或是利用體外移植β-cells做替換的方法，對治療第一型糖尿病的病患而言可說是一線曙光，然而，病患對β-cells的需求量遠大於目前所能提供的量，如果能夠發現胰臟內分泌細胞形成的機制，說不定能夠增進治療第一型糖尿病的治療方式；在先前的研究藉由differential screening的方式發現了一個尚未被鑑定的基因，將之命名為內胚層衍生激素 (EDF)，這個基因會轉譯出一個大約70KDa的蛋白質，並且在其蛋白質的N端帶有一段分泌型的信號

Beta cells are a type of cells located in the islets of Langerhans of pancreas. They make up 65-80% of the cells in the islets and produce insulin, a hormone that controls the level of glucose in the blood. It is known that type I diabetes results from the anatomical or functional loss of beta cells in pancreas. Regeneration or replacement of beta cells in diabetic patients is a very promising way to treat them. However, replacement of beta cells in diabetic patients is limited by the shortage of islet cells. If one can discover the mechanisms underlying formation of the endocrine pancreas, the development of such therapies will be accelerated. Previously, a novel gene, Endoderm Derived Factor (EDF), has been identified from a differential screening. This gene encodes a protein of ~70 kDa with a secretion signal peptide sequence at its N-terminus. When overexpressed in cultured cells, the EDF protein could be detected in the cultured medium, indicating that EDF is a secreted protein. Previous results also showed that EDF mRNA was expressed in pancreas and stomach from E9.5 to 11.5, and the expression was specifically restricted to the stomach in E12.5. Currently, our laboratory had generated anti-EDF antibody and several EDF transgenic mice line. We have generated transgenic mice in which the EDF coding sequence was placed under the control of the Pancreas Duodenum Homeobox-1 (PDX1) promoter. I have first confirmed the expression pattern of ectopic EDF was similar to the known expression pattern for PDX1. To further determine the functions of EDF during the development of pancreas, I analyzed the embryonic pancreas of EDF transgenic mice. I examined endocrine and exocrine cells using antibody staining against the acinar cell marker amylase and endocrine cell marker PTP-NP (protein tyrosine phosphatase - neural and pancreatic). However, I did not observe significant differences between EDF transgenic mice and their wild-type littermates in terms of the general morphology of their pancreas or the organization of the pancreatic endocrine and exocrine cells. I also did not detect alterations in glucose regulation in random-fed, fasted or glucose-challenged EDF transgenic mice. Further experiments are required to confirm the result. In the past, it was difficult to acquire large amounts of EDF recombinant protein. Therefore, I have generated an EDF fusion protein expression construct and utilized the recombinant Pichia strains (GS115, and KM71) to produce EDF fusion proteins. However, from yeast expression system, we obtained very little EDF protein. In mammalian cell system, we had improved the expression level of EDF protein with a modified expression protocol, and our result demonstrated that 5’UTR of EDF gene may play a role in regulating expression of EDF protein. And, I have generated recombinant bacmid contains the EDF gene and am ready to transfect insect cells to produce recombinant baculovirus.

Abstract (Chinese version)
Abstract (English version)
Chapter 1 Introduction and background……………………….......…1
1.1 Introduction to pancreatic structure……………………………………...…1
1.2 Diabetes mellitus……………………………………………………...…4
1.3 Morphological changes during pancreatic development…………………...…8
1.4 Signaling pathways and transcription factors involved in pancreas
development ………………………………………………………………9
1.5 Transcription factors important for pancreatic development …...……………10
1.6 Morphogen signaling in pancreas development……………………….……14
1.7 Research motive……………………………………………………..…17
Chapter 2 Materials and methods………………………………….…19
2.1 Gene expression constructs preparation………………………………...…19
2.2 In situ hybridization……………………………………………….……22
2.3 Recombinant protein expression (Yeast) ……………………………….…25
2.4 Generating EDF Recombinant bacmid (Baculovirus)………………………26
2.5 Cell Culture and transfection………………………………………….…26
2.6 Purification of EDF-myc/His Fusion Protein………………………………27
2.7 Immunoprecipitation……………………….………………………...…27
2.8 Western blot and coomassive blue staining……………………………...…28
2.9 Breeding, and genotyping of mice…………………………………….…29
2.10 Histology………………………………………………………..……29
2.11 Immunohistochemistry…………………………………………..……30
2.12 BrdU incorporation and histological staining…………………..…………30
2.13 Determination of blood glucose and Glucose tolerance testing………..……31
2.14 Animal models………………………………….………………..…31
Chapter 3 Result…………………………………………………….…32
3.1 Reconfirming the expression pattern of EDF in embryos of early developing
stages………………………………………………………………..……32
3.2 EDF is a secreted protein…………………………………………..……32
3.3 Ectopic EDF protein expression in pancreas of transgenic
mouse………………………………………………………………..…33
3.4 Blood glucose level of transgenic mice appear unaffected………………..…34
3.5 No significant differences in general morphology or organization of the pancreatic
cells between EDF transgenic mice and wild-type littermates……………...……34
3.6 Proliferation of the pancreatic cells between EDF transgenic mice and wild-type
littermates………………………………..…………………………...…35
3.7 Expression of Recombinant Pichia Strains……………………………...…35
3.8 Generating the EDF recombinant bacmid for recombinant protein expression
(Baculovirus)…………………………………………………………..…36
3.9 Expression of EDF protein in mammalian cell………………………...…36
Chapter 4 Discussion…………………………………………………..38
Chapter 5 Reference………………………………………………...…42
Chapter 6 Figures………………………………………………….…47
Appendix……………………………………………………………….60

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