The embryonic source and chemical nature of those factor/s directing the in vivo differentiation of melanocytes from crest cells are as yet unknown. To begin to address this issue, it is important to establish exactly when and where these signa/s first exert their effects. Therefore, in the present study, overtly differentiated melanocytes containing melanin were quantitated in developing Black Australorp X New Hampshire Red chick embryos. In contrast to previous studies, it was found that embryos synthesize melanin from as early as Day 5 of development, and that at this stage, the melanocytes are predominantly dermally located. Between 5 and 8 days, the numbers of both dermal and epidermal melanocytes increase, after which the dermal melanocyte population declines rapidly while the number of epidermal melanocytes continues to increase. These findings suggest that premelanocytes do not have to be epidermally located to initiate terminal differentiation and implicate the dermis as a possible source of melanocyte inducing factor/s. The next step was to examine stages of development prior to the onset of pigment production. For this reason, tyrosinase was purified for use as antigen in the production of a polyclonal antibody. The antibody was tested for specificity by western blotting, - immunocytochemistry and immunoinhibition procedures. Lack of specificity was demonstrated, rendering it unsuitable as an antibody marker for early melanocytes. Fowl melanocytes are thought to differentiate into either eumelanosome- or pheomelanosome synthesizing cells. To test the validity of this concept, embryonic skin of the red/black cross breed were screened for possible mixed type melanocytes by electron microscopy. The melanocytes contained melanosomes with a matrix of irregularly arranged filaments amongst typical eumelanogenic melanosomes. This suggests that these chick melanocytes may synthesize both eumelanosomes and pheomelanosomes in single cells. In a further study on pure breeding New Hampshire Reds, it was found that the melanocytes contained a mixture of typical and less typical pheomelanosomes. Outer membrane indentations in the latter melanosome type suggest that tyrosinase may enter these pheomelanosomes by a mechanism related to that proposed for the melanosomes of goldfish.

Early investigations of the urokinase type plasminogen activator (uPA) receptor (uPAR) and its ligand, uPA, were limited to their role in degradation of the extracellular matrix (ECM) and invasion. Emerging evidence revealed that uPAR and its relationship with uPA and/or transmembrane proteins, such as the integrins, affects cell-ECM adhesion events and proliferation. These events are tightly coordinated and essential for epithelial tissue development. However, unregulated expression of molecules involved in cell adhesion and proliferation plays a significant role in tumour development and metastasis. The overexpression of uPAR is linked to several cancer types, including human oesophageal squamous cell carcinoma (HOSCC). This study examines the contribution of uPAR, and its communication with extracellular components, to cell-ECM adhesion and/or proliferation of HOSCC cells. The confirmation of the uPAR and 1-integrin expression as well as uPA secretion in the HOSCC cells lines, established these lines as excellent models for further investigation. In all the HOSCC cell lines, uPAR associated with integrin-linked kinase, a scaffolding protein in cell-ECM adhesion events. Data presented in this investigation confirmed that the interaction of uPAR with uPA or 1-integrin contributed to adhesion of the HOSCC cell lines on collagen type I and vitronectin. It was clearly established that uPAR also played a part in the proliferation of all the HOSCC cell lines. The uPAR role in proliferation is influenced by: a) The absence or presence of collagen type I or vitronectin substrates; b) The activation of uPAR by endogenous uPA; c) The uPAR/1-integrin interaction; d) the presence of transforming growth factor  and epidermal growth factor. In the current study, it was successfully demonstrated that uPAR, and its relationship with the ECM and growth factors, contributes to adhesion and proliferation during the progression of HOSCC. This gives uPAR a considerable value as a therapeutic target for HOSCC.

No description available.

Ultra-microfluorometric techniques were adapted to follow several compounds related to energy metabolism through the developmental cycle of Dictyostelium discoideum. Each compound (ATP, trehalose, glucose, and ammonium ion) was found to be present in stalk and/or spore cells. The accumulation of NH₄⁺ was interpreted as an indication of protein degradation, a source of energy in this organism. During the early stages of differentiation NH₄⁺ was localized only in stalk cells. However, it accumulated in spore cells during culmination such that levels were comparable in the two cell types by the end of development. Trehalose, an energy source for germinating spores, was found in both cell types but was preferentially degraded in stalk cells late in development. Glucose, the degradation product of trehalose, was localized in stalk cells and varied inversely with trehalose in prestalk cells. ATP was not localized in a specific cell type during development. However, ATP declined in stalk cells at an earlier stage of development. These findings emphasize the need for knowledge of cell-specific events involved in the differentiation of this and other organisms. / Master of Science

No description available.

Li, Sui Mui. / On t.p. "beta" appears as the Greek letter. / Thesis submitted in: December 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 182-189). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vi / List of figures --- p.xii / List of appendices --- p.xix / Abbreviations --- p.xx / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Role of PPARP in adipocyte differentiation - an in vitro study --- p.20 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.23 / Chapter 2.2.1 --- Preparation ofPPARβ (+/+) and PPARβ (-/-) MEFs --- p.23 / Chapter 2.2.1.1 --- Materials --- p.23 / Chapter 2.2.1.2 --- Methods --- p.23 / Chapter 2.2.1.2.1 --- Isolation of MEFs --- p.23 / Chapter 2.2.1.2.2 --- Passage ofMEF culture --- p.25 / Chapter 2.2.2 --- Genotyping of PPARβ (+/+) and PPARβ (-/-) MEFs --- p.25 / Chapter 2.2.2.1 --- Materials --- p.26 / Chapter 2.2.2.2 --- Methods --- p.26 / Chapter 2.2.2.2.1 --- Primer design --- p.26 / Chapter 2.2.2.2.2 --- Genomic DNA extraction --- p.27 / Chapter 2.2.2.2.3 --- PCR reaction --- p.29 / Chapter 2.2.3 --- Western blotting of PPARβ(+/+) and PPARβ (-/-) MEFs --- p.30 / Chapter 2.2.3.1 --- Materials --- p.30 / Chapter 2.2.3.2 --- Methods --- p.31 / Chapter 2.2.3.2.1 --- Preparation of nuclear extracts --- p.31 / Chapter 2.2.3.2.2 --- Western blot --- p.32 / Chapter 2.2.4 --- Induction of adipocyte differentiation of PPARβ (+/+) and PPARβ(-/-) MEFs --- p.33 / Chapter 2.2.4.1 --- Materials --- p.34 / Chapter 2.2.4.2 --- Methods --- p.34 / Chapter 2.2.4.2.1 --- Seeding ofMEFs --- p.34 / Chapter 2.2.4.2.2 --- Adipocyte differentiation --- p.35 / Chapter 2.2.5 --- Oil Red O staining of differentiated PPARβ(+/+) and PPARβ(-/-) MEFs --- p.36 / Chapter 2.2.5.1 --- Materials --- p.36 / Chapter 2.2.5.2 --- Method --- p.37 / Chapter 2.2.5.2.1 --- Oil Red O staining --- p.37 / Chapter 2.2.6 --- Determination of triglyceride-protein assay of differentiated PPARβ (+/+) and PPARβ (-/-) MEFs --- p.37 / Chapter 2.2.6.1 --- Materials --- p.39 / Chapter 2.2.6.2 --- Methods --- p.39 / Chapter 2.2.6.2.1 --- Lysis of differentiated MEFs --- p.39 / Chapter 2.2.6.2.2 --- Measurement of triglyceride concentration in cell lysate --- p.40 / Chapter 2.2.6.2.3 --- Measurement of protein concentration in cell lysate --- p.41 / Chapter 2.2.7 --- Preparation of PPARβ(+/+) and PPARβ (-/-) MEF RNA for RT-PCR and Northern blot analysis --- p.42 / Chapter 2.2.7.1 --- Materials --- p.42 / Chapter 2.2.7.2 --- Method --- p.42 / Chapter 2.2.7.2.1 --- RNA isolation --- p.42 / Chapter 2.2.8 --- RT-PCR analysis of differentiated PPARβ(+/+) and PPARβ (-/-) MEFs --- p.44 / Chapter 2.2.8.1 --- Materials --- p.45 / Chapter 2.2.8.2 --- Methods --- p.45 / Chapter 2.2.8.2.1 --- Primer design --- p.45 / Chapter 2.2.8.2.2 --- RT-PCR --- p.46 / Chapter 2.2.9 --- Northern blot analysis of differentiated PPARβ(+/+) and PPARβ (-/-) MEFs --- p.47 / Chapter 2.2.9.1 --- Materials --- p.48 / Chapter 2.2.9.2 --- Methods --- p.49 / Chapter 2.2.9.2.1 --- Preparation of cDNA probes for Northern blotting --- p.49 / Chapter 2.2.9.2.1.1 --- RNA extraction --- p.49 / Chapter 2.2.9.2.1.2 --- Primer design --- p.49 / Chapter 2.2.9.2.1.3 --- RT-PCR of extracted mRNA --- p.50 / Chapter 2.2.9.2.1.4 --- Subcloning of amplified cDNA products --- p.50 / Chapter 2.2.9.2.1.5 --- Screening of recombinant clones by phenol-chloroform extraction --- p.51 / Chapter 2.2.9.2.1.6 --- Confirmation of the recombinant clones by restriction enzyme site mapping --- p.52 / Chapter 2.2.9.2.1.7 --- Confirmation of the recombinant clones by PCR method --- p.52 / Chapter 2.2.9.2.1.8 --- Mini-preparation of plasmid DNA from the selected recombinant clones --- p.54 / Chapter 2.2.9.2.1.9 --- Preparation of cDNA probes --- p.54 / Chapter 2.2.9.2.1.10 --- Formaldehyde agarose gel electrophoresis of RNA --- p.55 / Chapter 2.2.9.2.1.11 --- Hybridization and color development --- p.56 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Confirmation of PPARβ(+/+) and PPARβ (-/-) MEFs genotypes --- p.58 / Chapter 2.3.2 --- PPARβ (-/-) MEFs differentiated similarly to PPARβ(+/+) MEFs as measured by Oil Red O staining --- p.61 / Chapter 2.3.3 --- PPARβ (-/-) MEFs differentiated similarly to PPARβ(+/+) MEFs as reflected by their intracellular triglyceride contents --- p.64 / Chapter 2.3.4 --- PPARβ(-/-) MEFs expressed the adipocyte differentiation marker genes similarly to PPARβ (+/+) MEFs --- p.66 / Chapter 2.4 --- Discussion --- p.77 / Chapter Chapter 3 --- Role of PPARβ in adipocyte differentiation and lipid homeostasis - an in vivo study --- p.82 / Chapter 3.1 --- Introduction --- p.83 / Chapter 3.2 --- Materials and Methods --- p.85 / Chapter 3.2.1 --- Animal and high fat diet treatment --- p.85 / Chapter 3.2.1.1 --- Materials --- p.85 / Chapter 3.2.1.2 --- Method --- p.86 / Chapter 3.2.1.2.1 --- Animal treatment --- p.86 / Chapter 3.2.2 --- Tail-genotyping of PPARβ (+/+) and PPARβ (-/-) mice --- p.87 / Chapter 3.2.2.1 --- Materials --- p.87 / Chapter 3.2.2.2 --- Methods --- p.88 / Chapter 3.2.2.2.1 --- DNA extraction from tail --- p.88 / Chapter 3.2.2.2.2 --- PCR tail-genotyping --- p.89 / Chapter 3.2.3 --- "Measurement of serum triglyceride, cholesterol and glucose levels by enzymatic and spectrophometric methods" --- p.89 / Chapter 3.2.3.1 --- Materials --- p.90 / Chapter 3.2.3.2 --- Methods --- p.91 / Chapter 3.2.3.2.1 --- Serum preparation --- p.91 / Chapter 3.2.3.2.2 --- Measurement of serum triglycerides --- p.91 / Chapter 3.2.3.2.3 --- Measurement of serum cholesterol --- p.92 / Chapter 3.2.3.2.3 --- Measurement of serum glucose --- p.93 / Chapter 3.2.4 --- Measurement of serum insulin and leptin levels by ELISA --- p.94 / Chapter 3.2.4.1 --- Materials --- p.95 / Chapter 3.2.4.2 --- Methods --- p.95 / Chapter 3.2.4.2.1 --- Measurement of serum insulin --- p.95 / Chapter 3.2.4.2.2 --- Measurement of serum leptin --- p.97 / Chapter 3.2.5 --- "Histological studies of liver, interscapular BF and gonadal WF pads" --- p.99 / Chapter 3.2.5.1 --- Materials --- p.100 / Chapter 3.2.5.2 --- Methods --- p.100 / Chapter 3.2.5.2.1 --- "Fixation, dehydration, embedding in paraffin and sectioning" --- p.100 / Chapter 3.2.5.2.2 --- H&E staining --- p.101 / Chapter 3.2.6 --- Analyses of fecal lipid contents --- p.102 / Chapter 3.2.6.1 --- Materials --- p.102 / Chapter 3.2.6.2 --- Method --- p.103 / Chapter 3.2.6.2.1 --- Extraction of lipid contents from stools --- p.103 / Chapter 3.2.7 --- Statistical analysis --- p.104 / Chapter 3.3 --- Results --- p.105 / Chapter 3.3.1 --- Confirmation of genotypes by PCR --- p.105 / Chapter 3.3.2 --- PPARβ (-/-) mice were more resistant to high fat diet-induced obesity --- p.105 / Chapter 3.3.3 --- PPARβ (-/-) mice consumed similarly as to PPARβ (+/+) counterparts… --- p.122 / Chapter 3.3.4 --- Effect of high fat diet on organ weights --- p.128 / Chapter 3.3.4.1 --- PPARβ (-/-) mice were more resistant to high fat diet-induced liver hepatomegaly --- p.134 / Chapter 3.3.4.2 --- PPARβ (-/-) mice were resistant to high fat diet-induced increased white fat depots --- p.134 / Chapter 3.3.4.3 --- PPARβ (-/-) mice were resistant to high fat diet-induced increased brown fat mass --- p.137 / Chapter 3.3.5 --- Effect of high fat diet on organ histology --- p.142 / Chapter 3.3.5.1 --- PPARβ(-/-) mice were more resistant to high fat diet-induced liver steatosis --- p.143 / Chapter 3.3.5.2 --- No defect in white adipocyte expansion in PPARβ(-/-) mice upon high fat diet feeding --- p.153 / Chapter 3.3.5.3 --- No defect in brown adipocyte expansion in PPARβ (-/-) mice upon high fat diet feeding --- p.159 / Chapter 3.3.6 --- "Effect on high fat diet on serum cholesterol, triglyceride, glucose, insulin and leptin levels" --- p.164 / Chapter 3.3.6.1 --- "PPARβ (-/-) mice had a lower serum cholesterol level, but a similar triglyceride level as compared to PPARβ (+/+) mice upon high fat diet feeding" --- p.165 / Chapter 3.3.6.2 --- PPARβ (-/-) mice were resistant to high fat diet-induced insulin resistance --- p.167 / Chapter 3.3.6.3 --- PPARβ (-/-) mice had a similar serum leptin level as PPARβ (+/+) mice --- p.170 / Chapter 3.3.7 --- No decision made in fecal lipid content of PPARβ (+/+) and PPARβ (-/-) mice --- p.173 / Chapter 3.4 --- Discussion --- p.176 / References --- p.182 / Appendices --- p.190

Yung, Him Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vi / Publications based on work in this thesis --- p.vii / Abbreviations --- p.viii / Contents --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Objectives and overview of this study --- p.1 / Chapter 1.2 --- Rat pheochromocytoma (PC12) cells --- p.3 / Chapter 1.3 --- Prostanoids and their receptors --- p.4 / Chapter 1.4 --- Roles of prostanoids --- p.7 / Chapter 1.5 --- Nerve growth factor (NGF) and its receptors --- p.9 / Chapter 1.6 --- Change of gene expressions by NGF in PC12 cells --- p.10 / Chapter 1.7 --- Signaling pathways involved in NGF-induced differentiation of PC12 cells --- p.12 / Chapter 1.8 --- Classification of adenylyl cyclases --- p.14 / Chapter 1.9 --- Methods to study differentiation of PCI 2 cells --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.19 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.2 --- Cell culture medium and buffers --- p.25 / Chapter 2.3 --- Buffers and solutions for assay of [3H]inositoI phosphates ([3H]IP) production --- p.25 / Chapter 2.4 --- Buffers and solutions for assay of [3H]cAMP production --- p.27 / Chapter 2.5 --- Buffers and solutions for Western blotting --- p.28 / Chapter 2.6 --- Methods --- p.30 / Chapter 2.6.1 --- Maintenance of PC12 cells --- p.30 / Chapter 2.6.2 --- General culture condition of PCI2 cells for NGF treatment --- p.31 / Chapter 2.6.3 --- Determination of phospholipase C activity in PC12 cells --- p.31 / Chapter 2.6.3.1 --- Principle of assay --- p.31 / Chapter 2.6.3.2 --- Column preparation --- p.32 / Chapter 2.6.3.3 --- Measurement of [3H]IP production --- p.33 / Chapter 2.6.3.4 --- Data analysis --- p.34 / Chapter 2.6.4 --- Determination of adenylyl cyclase activity in PC12 cells --- p.35 / Chapter 2.6.4.1 --- Principle of assay --- p.35 / Chapter 2.6.4.2 --- Column preparation --- p.35 / Chapter 2.6.4.3 --- Measurement of [3H]cAMP production --- p.36 / Chapter 2.6.4.4 --- Data analysis --- p.37 / Chapter 2.6.5 --- Determination of neurofilament protein expression in PC12 cells by Western blotting --- p.38 / Chapter 2.6.6 --- Determination of adenylyl cyclase isoform expression in PC12 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) --- p.39 / Chapter 2.6.6.1 --- Isolation of total cellular RNA --- p.39 / Chapter 2.6.6.2 --- Synthesis of first strand cDNA by reverse transcription (RT) --- p.40 / Chapter 2.6.6.3 --- Polymerase Chain Reaction (PCR) --- p.41 / Chapter 2.6.6.4 --- Agarose gel electrophoresis --- p.41 / Chapter 2.6.7 --- Neurite quantification --- p.42 / Chapter 2.6.8 --- Trypan blue exclusion test --- p.42 / Chapter Chapter 3 --- Results --- p.45 / Chapter 3.1 --- Characterization of prostanoid receptor expression in PC12 cells . --- p.45 / Chapter 3.1.1 --- Study of the presence of Gq-coupled prostanoid receptors --- p.45 / Chapter 3.1.2 --- Study of the presence of Gs-co»pled prostanoid receptors --- p.47 / Chapter 3.1.3 --- Study of the presence of Gi-coupled prostanoid receptors --- p.48 / Chapter 3.1.4 --- Further proof of EP3 expression in PC12 cells --- p.50 / Chapter 3.1.5 --- Discussion --- p.51 / Chapter 3.2 --- Time course effect of NGF on PC12 cells --- p.65 / Chapter 3.2.1 --- Effect of NGF on PGE2-mediated inhibition of forskolin-stimulated [3H]cAMP production --- p.65 / Chapter 3.2.2 --- Effect of NGF on basal and forskolin-stimulated [3H]cAMP production --- p.67 / Chapter 3.2.3 --- Acute effect of NGF on [3H]cAMP production --- p.70 / Chapter 3.2.4 --- Effect of NGF withdrawal on basal and forskolin-stimulated [3H]cAMP production --- p.71 / Chapter 3.2.5 --- Effect of NGF on adenylyl cyclase gene expression --- p.72 / Chapter 3.2.6 --- Discussion --- p.74 / Chapter 3.3 --- Quantification of the degree of differentiation of PC12 cells --- p.89 / Chapter 3.3.1 --- Expression of neurofilament protein as a marker of differentiation --- p.89 / Chapter 3.3.2 --- Neurite assays --- p.90 / Chapter 3.3.2.1 --- Manual assessment of PC12 cells --- p.90 / Chapter 3.3.2.2 --- Quantification of images of PC1 2 cells --- p.91 / Chapter 3.3.3 --- Discussion --- p.93 / Chapter 3.4 --- Adenosine A2a receptor activity in PC12 cells --- p.106 / Chapter 3.4.1 --- Effect of NGF on A2Areceptor-mediated [3H]cAMP production --- p.106 / Chapter 3.4.2 --- Synergistic activation of adenylyl cyclase by A2A receptor and forskolin --- p.108 / Chapter 3.4.3 --- Chronic and acute effect of ADA and ZM241385 on [3H]cAMP production --- p.109 / Chapter 3.4.3.1 --- Chronic effect of ADA and ZM241385 --- p.110 / Chapter 3.4.3.2 --- Acute effect of ADA and ZM241385 --- p.111 / Chapter 3.4.4 --- Discussion --- p.112 / Chapter Chapter 4 --- Discussion and future perspectives --- p.121 / Chapter 4.1 --- Discussion --- p.121 / Chapter 4.2 --- Future perspectives --- p.131 / References --- p.133

Thesis (M.S.)--University of Missouri--Columbia, 2004. / Typescript. Includes bibliographical references (leaves 97-111). Also issued on the Internet.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18899号 / 医博第4010号 / 新制||医||1009(附属図書館) / 31850 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 三森 経世, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

胰臟內分泌細胞分化的調控事件的研究揭示了胰島素分泌細胞的形成。這一原理既有利於體外誘導用於移植的胰島素分泌細胞，又可應用于糖尿病病人自體胰島素分泌細胞的再生。正在發育的組織和器官中，發現了腎素血管緊張素（RAS）成員，揭示了他們在發育過程中的潛在調控作用。另外，對 RAS 信號系統做出應答的活性氧化物質（ROS），被認為是第二信使，通過對轉錄調控因子的氧化還原的修飾促進分化。作為 ROS 的主要來源，NADPH 已被證實在各類細胞和組織中參與了祖細胞的分化。儘管如此，依賴於 NADPH 氧化酶的 ROS對于胰腺內分泌細胞分化的調控作用仍不清楚。基於這個背景，本研究致力於揭示 RAS 和 NADPH 氧化酶依賴性 ROS 在胰腺內分泌細胞分化中的作用。本實驗將在小鼠胰臟原基培養物和尿鏈黴素（STZ）誘導的新生大鼠上進行。 結果顯示，經典 RAS 成員中的血管緊張素 2 型受體（AT₂R）分佈於內分泌祖細胞的細胞核，之後穿梭定位於胰島素分泌細胞的細胞質。阻斷 AT₂R 功能抑制了Ngn3，胰島素的表達以及 β 細胞的增值。在不同的胚胎期 ROS 的水平發生了改變。對于培養的胰臟原基施加適當的外源 ROS，刺激了內分泌細胞的分化。同時，ROS 清除劑減弱了胰島細胞分化和成熟的標記基因的表達。NOX4 以及其相關的亞基 p22phox 是 NADPH 氧化酶成員，其在胰臟發育過程中的變化同 ROS 水平的變化相似，並且持續表達與內分泌細胞系統。在 NGN3 高表達的胚胎期15.5 天，它們定位于表達 NGN3 的細胞；在 NGN3 表達下調，且胰島素表達升高的胚胎期 17.5 天，它們分佈於胰島素表達細胞。而且，NADPH 氧化酶的抑製劑 DPI 削弱了胰臟培養物中的內分泌祖細胞的分化, 外源 H₂O₂ 的加入扭轉了這一現象。 / 另一方面，在 STZ 誘導的新生大鼠的研究中，DPI 負調節 β 細胞的再生。血糖失調，胰島結構毀壞以及血清胰島素匱乏的現象發生在了 DPI 處理組。另外，DPI 減弱了 NGN3 的表達而並非 Ki67, 顯示 β 細胞的分化而並非增值對於 ROS 的刺激進行了應答。在體內和體外的實驗中，DPI 也抑制了 NGN3 的轉綠調控因子 SOX9 在胰腺祖細胞中的表達。有趣的是，過表達 SOX9 可以恢復 DPI 引發的對於 NGN3 的抑制 。結合以上數據，本研究顯示 NADPH 氧化酶依賴性ROS 誘導的信號通路參與了胰腺祖細胞到胰島素分泌細胞的分化。 / Investigations into the regulatory events that modulate pancreatic endocrine cell differentiation shed light on the generation of sufficient insulin-producing cells in vitro for transplantation or regeneration of β cells in patients with diabetes. The expression of renin-angiotensin system (RAS) components has been detected in development tissue and organs, implicating their regulatory role in developmental processes. On the other hand, reactive oxygen species (ROS) are responsive to RAS signaling pathways and act as second messengers to promote differentiation through redox modification of transcriptional factors essential for differentiation. As a major source of ROS, NADPH oxidase has been shown to participate in the progenitor differentiation in a variety of cells and tissues. Despite this finding, the role of NADPH oxidase-dependent ROS in regulating pancreatic endocrine cell differentiation remains ambiguous. Against this background, the study was aimed at elucidating the roles of RAS components and NADPH oxidase-derived ROS during differentiation of pancreatic endocrine cells using mouse pancreatic rudiments and streptozotocin-treated neonatal rats. / Results showed that angiotensin II type 2 receptor (AT₂R), a major component of the classical RAS, was localized within the nuclei of endocrine progenitors in the cultured pancreatic rudiments; following the differentiation of endocrine progenitors into insulin producing cells, it translocated to cytoplasm. Blockade of AT₂R impeded the expression of Ngn3 and insulin as well as proliferation of β-cells. In addition, the dynamic changes of ROS levels were found in mouse pancreata at different embryonic days, concomitant with induction of endocrine cell differentiation induced by modest exogenous ROS in pancreatic rudiment cultures. Moreover, scavenger of ROS diminished the expression of islet cell markers for differentiation and maturation. NOX4 and its associated subunit p22phox, which are the member of NADPH oxidase, exhibited similar changes of expression to that of ROS levels during pancreas development and persisted in the endocrine lineage; they were located in NGN3⁺ cells at E15.5 during the burst of NGN3 expression and then distributed in insulin⁺ cell at E17.5, the latter being the phase that has a decline in NGN3 expression with an increase of insulin. Furthermore, administration of NADPH oxidase inhibitor, diphenylene iodonium (DPI) attenuated the differentiation of endocrine progenitors in rudiment cultures, while exogenous ROS reversed this effect. / On the other hand, studies performed in streptozotocin-induced neonatal rats showed that β cell regeneration was negatively affected by DPI treatments; consistently, impaired blood glucose control, disturbed islet architecture and deficient serum insulin were observed in DPI-treated groups. In addition, DPI treatments blunted NGN3 expression, but not Ki67-labeling beta-cells, suggesting that differentiation beyond proliferation of β-cells was accountable in response to ROS stimulation. Administration of DPI also suppressed the levels of SOX9, a transcriptional regulator of NGN3, in pancreatic progenitor cells, as evidenced by both in vivo and in vitro studies. Interestingly, over-expression of SOX9 could restore the repression of NGN3 induced by DPI. Taken all these data together, our results indicate that NADPH oxidase-dependent ROS-induced signaling pathway is involved in the differentiation of pancreatic endocrine progenitors into insulin-producing β cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Juan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 171-205). / Abstracts also in Chinese.