臺灣博碩士論文加值系統

研究顯示胰臟發育需要許多的轉錄因子進行嚴謹的調控，像是PDX1、 Ptf1a和Ngn3。其中Ngn3是一個重要的鹼性-螺旋-環-螺旋(basic helix-loop-helix)轉錄因子參與在所有內分泌細胞的發育過程中，因此Ngn3所表現的前驅細胞也被視為胰島前驅細胞出現的象徵。
為了找出在胰臟發育的過程中會和Ngn3有交互作用的蛋白質並調控Ngn3下游基因的活性進而調節內分泌細胞的分化。因此實驗室之前利用酵母菌雙雜交系統(Yeast two-hybrid assay)，以Ngn3為釣餌(Bait protein)篩選老鼠胚胎10.5天的胰臟芽互補DNA基因庫(Mouse embryonic 10.5 pancreatic bud cDNA library)。
在密集的篩選完畢後，我們找到了可能會和Ngn3有交互作用的蛋白質- Zinc finger protein 668簡稱為Zfp668，擁有16個Cys2-His2(C2H2)型態的鋅指模組(Zinc finger motif)。
在本研究中，利用GST pull-down assay我已經證實了Ngn3和Zfp668之間有直接的交互作用，而接下來也利用了共同免疫沉澱(Co-Immunoprecipitation)證明了Ngn3和Zfp668蛋白在哺乳細胞中有交互作用關係的存在。接下來為了進一步分析Zfp668哪些鋅指模組對於和Ngn3的交互作用是必須的，一系列帶有GST的Zfp668短縮蛋白(truncate protein)也被純化出來，其中我發現到Zfp668胺基酸80~164和278~542是最小的片段去調節和Ngn3的交互作用。此外藉由
螢光酶報告基因檢測(luciferase reporter assay)也證實了Zfp668會影響Ngn3下游基因NeuroD的表現量。而利用慢病毒異位表現(Ectopic expression)Ngn3和Zfp668在PANC-1細胞中也可以得到和螢光酶報告基因檢測相同的結果。另外我也篩選出能穩定表達Ngn3或Zfp668的PANC-1細胞株，以利後續對於探討Zfp668的生物性功能。

Previous studies have shown that several transcription factors are critical regulators of pancreatic development, such as PDX1, Ptf1a and Ngn3. In above mentioned, Ngn3 is especially important as a bHLH transcription factors that involved in development of all endocrine cell lineages and has been designated as a marker of islet precursor cells.
To identify proteins which interact with Ngn3 and possibly modulate the activity of Ngn3 down-stream gene to regulate endocrine cell differentiation during pancreatic development. Previously, our laboratory has used yeast two-hybrid system to screen a mouse embryonic (E10.5) pancreatic bud cDNA library using Ngn3 as a bait. After an intensive screening, Zinc finger protein 668 (Zfp668), which contain 16 C2H2 type zinc finger motif, had been identified to interact with Ngn3.
In my study, I have verified that Ngn3 has a direct binding with Zfp668 through GST pull-down assay, and subsequently used co-immunoprecipitation to prove the Ngn3-Zfp668 interaction in mammalian cells. To further dissect Zfp668 for motifs responsible for interacting with Ngn3, a series of GST-tagged Zfp668 truncate protein were purified. I found that the 80~164 and 278~542 amino acid region of Zfp668 are the shortest fraction to mediate the interaction with Ngn3. Significantly, I have also validated that Zfp668 could affect the expression of Ngn3 down-stream target (NeuroD) by luciferase reporter assay. Ectopic expression of Ngn3 and Zfp668 in PANC-1(human pancreatic carcinoma) cells with lentivirus also lead to the same conclusion. In addition, the stable PANC-1 cell line with Ngn3 or Zfp668 overexpression clone were established to determine the biological function of Zfp668.

中文摘要...................................................................................................................... I
ABSTRACT................................................................................................................II
第一章、前言 (INTRODUCTION AND BACKGOROUND)...............................-1-
1-1 胰臟 (Pancreas)的結構及生理功能...................................................................-1-
1-2 糖尿病 (Diabetes mellitus)….............................................................................-3-
1-3 胰臟的發育.........................................................................................................-6-
1-4 胰臟發育時期重要的轉錄因子 (Transcription factors) ..................................-7-
1-5 研究動機 (Research motive)............................................................................-10-
第二章、材料與方法 (MATERIALS AND METHODS).................................... -12-
2-1 材料部分...........................................................................................................-12-
2-2 方法部分...........................................................................................................-20-
2-2-1 質體建構...................................................................................................-20-
2-2-2 基因重組蛋白質表達及純化...................................................................-22-
小量誘導蛋白質表現............................................................................-22-
大量誘導蛋白質表現以及萃取蛋白質................................................-22-
GST融合蛋白的純化............................................................................-23-
HIS融合蛋白的純化.............................................................................-24-
GST及HIS融合蛋白的透析及濃縮.....................................................-24-
BSA蛋白質粗定量...............................................................................-25-
SDS-PAGE電泳分析...........................................................................-25-
SDS-PAGE膠體的染色與褪染............................................................-25-
SDS-PAGE膠體的乾燥保存................................................................-26-
西方墨點法 (Western blotting)............................................................-26-
In vitro GST-fusion protein pull-down assay.........................................-27-
2-2-3 細胞實驗操作..........................................................................................-27-
細胞繼代培養操作................................................................................-27-
細胞冷凍保存........................................................................................-28-
冷凍細胞活化........................................................................................-28-
細胞株的質體短暫植入 (Transient transfection)................................-28-
細胞株蛋白質萃取................................................................................-29-
共同免疫沉澱 (Co-Immunoprecipitation with ANTI-FLAG M2 Affinity Gel)...-29-
共同免疫沉澱 (Co-Immunoprecipitation with Protein A SepharoseTM CL-4B)..-30-
慢病毒的製備........................................................................................-31-
慢病毒的感染與濃縮............................................................................-31-
以限定細胞培養液誘導PANC-1轉分化(Trans-differentiation)..........-32-
穩定細胞株篩選....................................................................................-32-
RNA萃取和反轉錄-聚合酶鏈鎖反應(Reverse transcriptase polymerase
chain reaction；RT-PCR)......................................................................-33-
第三章、實驗結果 (RESULT) .............................................................................-34-
3-1 利用Zfp668融合蛋白製備Zfp668多株抗體 ...............................................-34-
3-2 Zfp668多株抗體測試.....................................................................................-35-
3-3 Ngn3與Zfp668在HEK293T細胞中有交互作用的存在...............................-36-
3-4 利用細菌系統表現Ngn3及Zfp668融合蛋白...............................................-37-
3-5 Ngn3與Zfp668在試管中有交互作用的存在................................................-38-
3-6 異位表達Ngn3和Zfp668促使NeuroD的表現...............................................-39-
3-7 在PANC-1細胞中篩選出能穩定表達Ngn3或Zfp668的stable clone...........-40-
第四章、討論 (DISCUSSION) ............................................................................ -41-
4-1 蛋白質間交互作用.........................................................................................-41-
4-2 Zfp668對於內分泌細胞分化的影響.............................................................-42-
第五章、參考資料 (REFERENCES).......................................................................-45-
第六章、圖 (FIGURES) ..........................................................................................-49-
第七章、附件 (APPENDIX) ...................................................................................-59-

Ahlgren U, Jonsson J, Jonsson L, Simu K, Edlund H. (1998) Beta-cell-specific inactivation of the mouse Ipf1/ Pdx1 gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes Dev. 12, 1763–68

Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. (2001) Insulin production by human embryonic stem cells. Diabetes 50(8), 1691-7

Buchanan TA, Xiang A, Kjos SL, Lee WP, Trigo E, Nader I, Bergner EA, Palmer JP and Peters RK. (1998) Gestational diabetes: antepartum characteristics that predict postpartum glucose intolerance and type 2 diabetes in Latino women. Diabetes 47, 1302-10

Chae JH, Stein GH & Lee JE. (2004) NeuroD: the predicted and the surprising. Molecules and Cells 18, 271–88

Gauthier BR, Schwitzgebel VM, Zaiko M, Mamin A, Ritz-Laser B, Philippe J (2002) Hepatic nuclear factor-3 (HNF-3 or Foxa2) regulates glucagon gene transcription by binding to the G1 and G2 promoter elements. Mol Endocrinol 16(1), 170-83

Gradwohl G, Dierich A, LeMeur M, Guillemot F. (2000) Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proceedings of the National Academy of Sciences of the United States of America 97(4), 1607-11

Grapin-Botton A, Majithia AR, Melton DA. (2001) Key events of pancreas formation are triggered in gut endoderm by ectopic expression of pancreatic regulatory genes. Genes Dev 15(4), 444-54

Gu G, Dubauskaite J, Melton DA. (2002) Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129(10), 2447-57

Habener JF, Kemp DM, Thomas MK. (2005) Minireview: Transcriptional regulation in pancreatic development. Endocrinology 146(3), 1025–34
Haumaitre C, Lenoir O, Scharfmann R. (2008) Histone deacetylase inhibitors modify pancreatic cell fate determination and amplify endocrine progenitors. Mol Cell Biol 28(20), 6373-83

Heremans Y, Van De Casteele M, in't Veld P, Gradwohl G, Serup P, Madsen O, Pipeleers D, Heimberg H. (2002) Recapitulation of embryonic neuroendocrine differentiation in adult human pancreatic duct cells expressing neurogenin 3. J Cell Biol 159(2), 303-12

Holland AM, Hale MA, Kagami H, Hammer RE, MacDonald RJ. (2002) Experimental control of pancreatic development and maintenance. Proc Natl Acad Sci USA 99, 12236–41

Huang HP, Liu M, El-Hodiri HM, Chu K, Jamrich M, Tsai MJ. (2000) Regulation of the pancreatic islet-specific gene BETA2 (neuroD) by neurogenin 3. Mol Cell Biol 20, 3292–307

Isenman L, Liebow C, Rothman S. (1999) The endocrine secretion of mammalian digestive enzymes by exocrine glands. Am J Physiol 276(2 Pt 1), E223-32

Jonsson, J., Carlsson, L., Edlund, T., Edlund, H. (1994) Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371, 606–09

Kawaguchi Y, Cooper B, Gannon M, Ray M, MacDonald RJ, Wright CVE. (2002) The role of the transcriptional regulator PTF1-p48 in converting intestinal to pancreatic progenitors. Nat Genet 32, in press

Krapp A, Knofler M, Frutiger F, Hughes GJ, Hagenbuchle O, Wellauer PK. (1996) The p48 DNA-binding subunit of transcription factor PTF1 is a new exocrine pancreas-specific basic helix-loop-helix protein. EMBO J 15, 4317–29

Krapp A, Knofler M, Ledermann B, Burki K, Berney C, Zoerkler N, Hagenbuchle O, Wellauer PK. (1998) The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas. Genes Dev. 12, 3752–63

Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, Young H, Richardson M, Smart NG, Cunningham J, Agulnick AD, D'Amour KA, Carpenter MK, Baetge EE. (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol 26(4), 443-52

Lefebvre B, Belaich S, Longue J, Vandewalle B, Oberholzer J, Gmyr V, Pattou F, Kerr-conte J. (2010) 5’-AZA induces Ngn3 expression and endocrine differentiation in the PANC-1 human ductal cell line. BBRC 391, 305–9

Malecki MT, Jhala US, Antonellis A, Fields L, Doria A, Orban T, Saad M, Warram JH, Montminy M and Krolewski AS. (1999) Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus. Nature Genetics 23, 323–28

McKinnon CM, Docherty K. (2001) Pancreatic duodenal homeobox-1, PDX-1, a
major regulator of beta cell identity and function. Diabetologia 44, 1203–14

Misra SP, Dwivedi M. (1990) Pancreaticobiliary ductal union. Gut 31(10), 1144-49.

Naya FJ, Huang HP, Qiu Y, Mutoh H, DeMayo FJ, Leiter AB and Tsai MJ. (1997) Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroDdeficient mice. Genes Dev. 11, 2323–34

Naya FJ, Stellrecht CM, Tsai MJ. (1995) Tissue-specific regulation of the insulin gene by a novel basic helix–loop–helix transcription factor. Genes Dev. 9, 1009–19

Ogihara T, Fujitani Y, Uchida T, Kanno R, Choi JB, Hirose T, Kawamori R, Watada H. (2008) Combined expression of transcription factors induces AR42J-B13 cells to differentiate into insulin-producing cells. Endocr J. 55(4):691-8

Owens MD, Kieffer EC, Chowdhury FM. (2006) Preconception care and women with or at risk for diabetes: implications for community intervention. Matern Child Health J 10, S137-41

Rother KI. (2007) Diabetes treatment--bridging the divide. N Engl J Med 356, 1499-501

Rukstalis JM, Habener JF. (2009) A master regulator of pancreatic islet differentiation and regeneration. Islets 1:3, 177-84
Segev H, Fishman B, Ziskind A, Shulman M, Itskovitz-Eldor J. (2004) Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 22(3), 265-74

Slack, JM. (1995) Developmental biology of the pancreas. Development 121, 1569-80

Smith SB, Gasa R, Watada H, Wang J, Griffen SC, German MS. (2003) Neurogenin3 and hepatic nuclear factor 1 cooperate in activating pancreatic expression of Pax4. J Biol Chem 278, 38254-9

Stoffers DA, Ferrer J, Clarke WL, Habener JF. (1997) Early-onset type-II diabetes mellitus (MODY4) linked to IPF1. Nat Genet 17, 138–39

Vinik AI, Fishwick DT and Pittenger G. (2004) Advances in diabetes for the
millennium: toward a cure for diabetes. MedGenMed 6, 12

Wang S, Jensen JN, Seymour PA, Hsu W, Dor Y, Sander M, Magnuson MA, Serup P, Gu G. (2009) Sustained Neurog3 expression in hormone-expressing islet cells is required for endocrine maturation and function. Proc Natl Acad Sci U S A 106(24), 9715-20

Wierup N, Svensson H, Mulder H, Sundler F (2002) The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas. Regul. Pept. 107, 63–69

White P, May CL, Lamounier RN, Brestelli JE, Kaestner KH. (2008) Defining pancreatic endocrine precursors and their descendants. Diabetes 57(3), 654-68

Zimmet P, Alberti KG and Shaw J. (2001) Global and societal implications of the diabetes epidemic. Nature 414, 782-87

Zhang T, Wang H, Saunee NA, Breslin MB, Lan MS. (2010) Insulinoma-Associated Antigen-1 Zinc-Finger Transcription Factor Promotes Pancreatic Duct Cell Trans-Differentiation. Endocrinology 151(5), 2030–39