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The Pancreas Is a Mixed Exocrine and Endocrine Gland Organ

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The Pancreas Is a Mixed Exocrine and Endocrine Gland Organ SSStttooonnnyyy BBBrrrooooookkk UUUnnniiivvveeerrrsssiiitttyyy The official electronic file of this thesis or dissertation is maintained by the University Libraries on behalf of The Graduate School at Stony Brook University. ©©© AAAllllll RRRiiiggghhhtttsss RRReeessseeerrrvvveeeddd bbbyyy AAAuuuttthhhooorrr... Pancreas specific ablation of 1 integrin in mouse: Biology of epithelial cells in a model of chronic pancreatitis. A Dissertation presented By Lorenzo Bombardelli To The Graduate School In Partial Fullfillment of the Requirements For the Degree of Doctor of Philosophy in Genetics Stony Brook University May 2008 Stony Brook University The Graduate School Lorenzo Bombardelli We, the dissertation committee for the above candidate for the Doctor of Philosophy degree, hereby recommend acceptance of this dissertation. Howard Crawford, Ph.D. Assistant Professor Dissertation Advisor Department of Pharmacological Sciences Daniel Bogenhagen, M.D. Professor Chairperson Department of Pharmacological Sciences Senthil Muthuswamy, Ph.D. Associate Professor Cold Spring Harbor Laboratory Kenneth Marcu, Ph.D. Professor Department of Biochemistry and Cell Biology Stanley Zucker, M.D. Professor School of Medicine This dissertation is accepted by the Graduate School Lawrence Martin Dean of the Graduate School ii Abstract of the Dissertation Pancreas specific ablation of 1 integrin in mouse: Biology of epithelial cells in a model of chronic pancreatitis. By Lorenzo Bombardelli Doctor of Philosophy in Genetics Stony Brook University 2008 Integrins are a large family of transmembrane molecules that mediate cell contact with the extracellular environment, especially the extracellular matrix. Integrin-mediated contact with the basement membrane underlying glandular epithelial cells, such as pancreatic acini, determines cellular polarity required for appropriate exocrine function. Growing evidence from in vivo and in vitro studies highlights the requirement of 1 integrin function in stem cells and in three-dimensional tumor biology, where the 1 integrin modulates cellular differentiation and contributes to the malignant behavior of cancer cells. I tested hypothesis that 1 integrin ablation in the pancreas would eliminate the ability of acinar cells to maintain contact with the basement membrane, potentially compromising their function in both normal and pathological situations. In order to address these questions and understand the fate of acinar cells lacking BM contact, a pancreas-specific knockout of integrin was developed. This allowed us to follow completely the biology of acinar cells, from development to adulthood, in normal condition and in the context of experimental pancreatitis. In my studies, I found that 1 integrin was not required for the initial establishment of acinar cell polarity, but was necessary for the maintenance of acinar iii homeostasis. I showed that integrin is involved in directional secretion, and this is a critical element for the maintenance of acinar cell integrity. Finally, I suggest the possibility that the contribution of integrin might be related to its signaling function, rather than to the role of physical cell-cell and cell-BM link. Accordingly, identifying the signaling defect that lead null mice to irreversible degeneration, could lead the way for the development of therapy of acute and chronic pancreatitis iv Table of contents List of figures .............................................................................................................. vii List of tables ............................................................................................................... viii Chapter 1 General Introduction ....................................................................................................... 1 Activation of integrins .............................................................................................. 12 Experimental models of pancreatitis ........................................................................... 6 Genetic and experimental evidence ............................................................................ 7 Integrin signaling ...................................................................................................... 13 Integrins .................................................................................................................... 10 Intracellular origin of pancreatitis ............................................................................... 7 Introduction to the pancreas ........................................................................................ 2 Pancreatic ablation of 1 integrin project description and rationale ........................................................................... 15 Pancreatic diseases ...................................................................................................... 4 Pathophysiology of pancreatitis .................................................................................. 5 Chapter 2 Experimental Procedures .............................................................................................. 17 Acinar cell preparation .............................................................................................. 18 Amylase activity assay and protein quantitiation. .................................................... 20 Experimental Pancreatitis and tissue processing ...................................................... 19 Genotyping and detection of floxed alleles recombination ....................................... 18 Immunofluorescence on isolated acini...................................................................... 20 Immunohistochemistry, Immunofluorescence and LacZ staining ............................ 19 Invasion assays.......................................................................................................... 22 Live cell imaging ...................................................................................................... 21 Methionine starvation ............................................................................................... 22 Migration assays ....................................................................................................... 22 MMP-7 RNAi silencing vector construction. ........................................................... 21 Pancreas specific ablation of the 1 integrin subunit ............................................... 18 Pancreatic tumorigenesis and progression in wild type and MMP-7-/- backgrounds. 23 Proliferation assays ................................................................................................... 21 Statistical Analysis .................................................................................................... 24 Tumorigenesis in athymic nude mice ....................................................................... 23 Chapter 3 Pancreatic ablation of 1 integrin results ........................................................................................................................ 26 Amylase secretion of integrin flox/flox Ptf1a-cre isolated acini .......................... 39 Cre-mediated recombination and pancreatic ablation of 1 integrin .................... 27 Loss of integrin results in pancreatic degeneration. ........................................ 29 Morphology of integrin flox/flox Ptf1a-cre isolated acini .................................... 32 Integrin flox/flox Ptf1a-cre mice have increased susceptibility to cerulein-induced pancreatitis and have a delayed recovery.......................................................... 37 v Chapter 4 Pancreatic ablation of 1 integrin Discussion ................................................................................................................. 46 Chapter 5 Matrix Metalloproteinase-7 in pancreatic cancer Animal models of pancreatic cancer ......................................................................... 56 Clinical studies .......................................................................................................... 62 Experimental design.................................................................................................. 54 In vitro studies........................................................................................................... 62 In vivo studies ........................................................................................................... 62 Introduction to pancreatitis and pancreatic cancer .................................................... 55 Matrix metalloproteinases and MMP-7 .................................................................... 59 MMP-7 Substrates .................................................................................................... 60 Pancreatic ductal adenocarcinoma ............................................................................ 55 Project description and rationale ............................................................................... 54 Role of acinar to ductal metaplasia ........................................................................... 59 Matrix Metalloproteinase-7 in pancreatic cancer ......................................................... 53 Chapter 6 Matrix Metalloproteinase-7 in pancreatic cancer Results ....................................................................................................................... 63 Invasion and migration assays .............................................................................
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