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Early pancreatectomy experiments performed in ducks and pigeons at the end of the 19th century revealed that avians, unlike mammals, do not display signs of diabetes. Relative to mammals, birds are considered hyperglycemic, displaying fasting blood glucose concentrations twice that of a normal human. While circulating levels of insulin are similar in avians and mammals, and structure and function of the insulin receptor are also conserved among vertebrate species, birds do not experience deleterious effects of chronic hyperglycemia as observed in mammals. Understanding avian glucose homeostasis, particularly in chickens, has both agricultural and biomedical implications. Improvement of feed efficiency and accelerated growth in poultry may come from a greater understanding of the physiological processes associated with glucose utilization in muscle and fat. The chicken has also recently been recognized as an attractive model for human diabetes, where there is a great need for preventative and therapeutic strategies. The link between type 2 diabetes and obesity, coupled with the inherent hyperglycemic nature of chickens, make chickens artificially selected for juvenile low (LWS) and high (HWS) body weight a favorable model for investigating glucose regulation and pancreas physiology. Oral glucose tolerance and insulin sensitivity tests revealed differences in threshold sensitivity to insulin and glucose clearance rate between the lines. Results from real-time PCR showed greater pancreatic mRNA expression of four glucose regulatory genes (preproinsulin, PPI; preproglucagon, PPG; glucose transporter 2, GLUT2; and pancreatic duodenal homeobox 1, Pdx1) in LWS, than HWS chickens. Histological analysis of pancreas revealed that HWS chickens have larger pancreatic islets, less pancreatic islet mass, and more pancreatic inflammation than LWS chickens, all of which presumably contribute to impaired glucose metabolism. In summary, results suggest that at selection age, there are differences in pancreas physiology that may explain the differences in glucose regulation between LWS and HWS. These data pave the way for future studies aimed at understanding the developmental regulation of endocrine pancreas function in chickens, as well as how aging affects homeostatic control of blood glucose in chickens. / Ph. D.

The pancreas is a secretory organ composed of exocrine and endocrine compartments. During development, endocrine cells delaminate from the pancreatic epithelium to associate with the local vasculature where they will secrete hormones such as insulin into the blood stream. The exocrine pancreas is composed of ductal cells which form a network of tubes to secrete and transport fluid carrying the digestive enzymes secreted from acini located at the terminal ends of ductal branches. Unlike many branched epithelia, the pancreas does not exhibit a stereotypical branching pattern. The ductal network develops from a mesh of interconnected lumens which are eventually pruned and give rise to a final tree-like structure optimized for the most economical delivery of enzymes and fluid to the digestive tract. In silico modelling suggests that fluid flow plays a role in resolving the final structure of the ductal network during development, indicating that physical forces may play a role in this self-organization Recent work in the adult human pancreas has shown that the cells of the small ducts in the most distal parts of the ductal network do not express the same transcripts as the proximal large ducts. The pancreas derives this structure and function from the differentiation and self-organization of progenitors into terminally differentiated cells which, together with mesenchymal cells and vasculature, contribute to the tissue niche of the organ. Despite the importance of this process in development and disease, little is known about how pancreatic progenitors balance differentiation with morphogenesis. The goal of this project was to uncover niche components that influence the differentiation of pancreas progenitors, and understand how identity and morphogenesis are mediated by niche-driven changes in gene expression. This remains a challenging process to understand due to limited accessibility of the embryonic pancreas. Therefore, human sphere and organoid models represent a valuable tool to address this question and were used together with expression profiling and manipulation of the extracellular environment to understand this relationship during pancreas development. Time-course bulk RNA sequencing of human pancreas progenitor spheres at different days of culture revealed the sequential processes happening as the cells form their niche, and then start proliferating and forming lumen. Notably, at the stage of lumen expansion, we observed an upregulation of genes associated with ductal epithelia, such as CFTR, MUC1, MUC6, and CA2 in tandem with increased expression of genes encoding proteins for ion and fluid secretion. This suggested hydraulics may act to integrate ductal differentiation with luminogenesis, which is consistent with in silico modelling and the secretory function of the pancreatic epithelia. Indeed, driving chloride ion secretion with the CFTR activator forskolin resulted in inflation of the sphere lumen and increased the expression of the ductal genes identified above. Induction of CFTR and MUC1 can also be achieved by inflating the lumen in a CFTR-independent manner using prostaglandin E2. This revealed the changes in gene expression were not due to a small feedback loop under the control of CFTR, and maybe due to morphological changes related to lumen inflation. Importantly, it was revealed that the induction of Cftr expression upon lumen inflation also occurred in pancreas explants isolated from embryonic mice, which suggests the relationship between lumen inflation and ductal identity is conserved between mouse and human. Datamining of single cell RNA sequencing of adult and fetal pancreas samples identified novel marker genes for progenitor, acinar, and large- and small-ducts of the human fetal pancreas. Comparison of these marker genes with gene expression patterns of pancreas progenitor spheres revealed a shift to a small-duct-like identity when the lumen is inflated. This shift seemed to be dependent on inflation of the lumen rather than cAMP signalling, as it is not observed in pancreas progenitors grown in 2D and treated with forskolin. The above experiments suggest a link between lumen inflation and small duct identity but the exact mechanism remains unclear. Lumen inflation is likely driven by an increase in hydrostatic pressure that occurs downstream of changes in osmotic pressure due to ion channel activation. Independent manipulation of osmotic pressure, hydraulic pressure, tissue stretching, and fluid shear stress will be valuable to decipher the mechanisms of ductal gene regulation. Taken together these results support the hypothesis that differential gene expression in ducts of different sizes is regulated by mechanical forces in the pancreas, and 3D sphere culture represents a powerful model to investigate these processes in finer detail.

Fetal pancreatic tissues can be a promising source for pancreatic progenitor cells (PPCs). In this regard, we have successfully isolated and characterized a population of fetal PPCs from first trimester human fetal pancreas using a previously established basic protocol. Upon exposure to a cocktail of conventional growth factors, these PPCs are amenable to differentiate into insulin-secreting islet-like cell clusters (ICCs); however, these ICCs have yet to exert additional efforts to direct to glucose-responsive cells. To address this issue, we have proposed two novel morphogenic factors in the present study, namely PDZ-domain-containing 2 (PDZD2) and angiotensin II (Ang II), a physiologically active peptide of the renin-angiotensin system (RAS), that potentially promote the differentiation and maturation of PPCs/ICCs. / In light of these findings, we conclude that we have discovered two novel mechanisms, the PDZD2 and Ang II/AT2 receptor signaling pathways, in the regulation of the development of PPCs/ICCs, thus implying their novel roles during islet development in vivo. The present study provides a "proof-of-principle" that a local RAS is critically involved in governing islet cell development. This work may contribute to devising protocols for maturation of pancreatic progenitors for clinical islet transplantation. / Local RASs have been reported to regulate the differentiation of tissue progenitor cells. It has yet to be confirmed whether such systems exist and govern the PPC development. To address this issue, we herein provided evidence that expression of RAS components was highly regulated throughout PPC differentiation. Locally generated Ang II was found to maintain PPC growth and differentiation via mediation of the Ang II type 1 and type 2 (AT1 and AT 2) receptors. We found that the AT2, but not AT1, receptor was a key mediator of Ang II-induced upregulation of beta-cell transcription factors. Transplantation of AT2 receptor-depleted ICCs into immune-privileged diabetic mice failed to ameliorate hyperglycemia, implying that AT2 receptors are indispensable during ICC maturation in vivo. / PDZD2 and its secreted form (sPDZD2) have been found to express in our fetal PPCs. We first evaluated the potential role of sPDZD2 in stimulating PPC differentiation and established an optimal concentration for such stimulation. We found that 10-9 M sPDZD2 promoted PPC differentiation, as evidenced by the up-regulation of the pancreatic endocrine markers and C-peptide content in the ICCs. It enhanced their expression of the L-type voltage-gated calcium ion channel (Cav1.2) and conferred an ability to secrete insulin in response to membrane depolarization. Yet these ICCs remained glucose-unresponsive because of the minimal expression of GLUT-2. We thus attempted to study another potential morphogenic candidate, Ang II. / To further test whether a functional RAS is present and if so, whether it regulates islet development in vivo, we employed a mouse embryo model at different embryonic days and reported a stronger AT2 receptor expression during the 2nd developmental transition of pancreas development. AT2 receptor blockade from e8.0 resulted in abnormalities in fetal pancreatic development. Neonates from these mother mice displayed destructed pancreas/islet architecture, a hampered ability in glucose-stimulated insulin-secretion possibly attributed to a decreased ratio of beta-cell to alpha-cell, and an impaired glucose tolerance at 4-wk old. / Leung, Kwan Keung. / Adviser: Po Sing Leung. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 254-284). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Implanted tissue engineered substitutes constitute dynamic systems, with remodeling mediated by both the implanted cells and the host. Thus, there exists a significant need for methods to monitor the function and morphology of tissue engineered constructs. Noninvasive monitoring using 1H Nuclear Magnetic Resonance (NMR) spectroscopy and imaging can prove to be the solution to this problem. Spectroscopy allows for assessment of cellular function through the monitoring of inherent metabolic markers, such as total-choline, while high resolution imaging enables the evaluation of construct morphology and interfacial remodeling. We applied these 1H NMR methods to monitor betaTC3 mouse insulinoma cells within hydrogel-based materials as a model pancreatic tissue substitute. In vitro research established a strong correlation between total-choline, measured by 1H NMR spectroscopy, and viable betaTC3 cell number, measured by MTT. Extending these methods to in vivo monitoring, however, was met with additional challenges. First, the implanted cells needed to be contained within a planar construct above a threshold density to allow for adequate quantification of the total-choline peak. Secondly, cell-free buffer zones between the implanted cells and the host tissue needed to be incorporated to prevent host tissue signal contamination. Finally, quantitative techniques needed to be developed to accurately account for contaminating signal from diffusing molecules. To overcome these challenges, a disk-shaped agarose construct, initially containing a minimum of 4 million betaTC3 cells and coated with an outer layer of pure agarose, was fabricated. Mathematical simulations aided the implant design by characterizing diffusive transport of nutrients and metabolites into and out of the construct. In vivo 1H NMR studies of these constructs implanted in mice established a strong correlation between total-choline, measured noninvasively using 1H NMR spectroscopy, and viable cell number, measured invasively using MTT. This study establishes total-choline as a reliable marker for noninvasively quantifying dynamic changes in viable betaTC3 cell number in vivo. 1H NMR imaging was used to monitor the implants structural integrity over time, while also assessing the hosts fibrotic response. We expect these studies to establish quantitative criteria for the capabilities and limitations of NMR methodologies for monitoring encapsulated insulinomas, as well as other tissue implants.

The proposal of this work was to determine the contribution of functional MR imaging techniques, i.e. secretin-enhanced MRCP (S-MRCP) and dynamic contrast-enhanced magnetic resonance (DCE-MR) imaging in the quantitative assessment of exocrine pancreatic function and perfusion.<p><p>The pancreas is both an exocrine and endocrine organ, though the exocrine tissue accounts for more than 90%. The exocrine pancreas is specialized in the synthesis and storage of digestive enzymes and in bicarbonate and water secretion in response respectively to various secretagogues (CCK, ach, GRP, VIP,…) and to secretin. <p>The arterial supply of the pancreas derives from branches of the celiac trunk and of the superior mesenteric artery. The microvascularity of the exocrine and the endocrine parts of the gland are anatomically and functionally separated, with differentially regulated blood perfusion. <p>Based on the knowledge of a close relationship between the activity of the gland and its blood supply, in normal conditions pancreatic perfusion responds to the functional state of the exocrine parenchyma: increased demands for exocrine secretions are associated with increased pancreatic blood flow. <p>The pancreatic gland can be involved at different degrees of severity in acute and chronic inflammatory processes due to various causes. In both processes microcirculatory changes occur and the pancreatic exocrine function can be impaired. Moreover, an exiguous microvascular component characterizes pancreatic ductal adenocarcinoma (PDA) related to a prominent stroma.<p><p><p>In the first section of this thesis, quantitative assessment of the pancreatic exocrine secretions was performed with S-MRCP in physiologic and non-physiologic conditions. The stimulating effect of secretin as well as the inhibitory effect of somatostatin on normal pancreas, both administered at different dose-regimens, were tested. The results of these investigations showed that quantitative S-MRCP is able to detect changes in pancreatic exocrine secretions correlated to the degree of stimulation or inhibition. <p>In pathologic settings, pancreatic exocrine secretions were assessed in chronic pancreatitis patients showing different degrees of severity, before and after endoscopic pancreatic duct drainage procedures (PDDP). In the group of patients presenting a reduced pancreatic exocrine reserve before treatment, quantitative S-MRCP showed a short-term improvement after PDDP. <p><p>In the second section, the feasibility and the reproducibility of DCE-MR imaging to quantify regional pancreatic perfusion was firstly investigated. DCE-MR imaging was performed in normal volunteers. Reference values for regional pancreatic perfusion were achieved with an intra-individual variability of 21%.<p>DCE-MR investigations were repeated during secretin stimulation and disclosed a significant increase of regional pancreatic perfusion in all individuals. <p>Secondly, DCE-MR imaging investigated benign and malignant focal pancreatic solid lesions and non tumoral tissue in patients undergoing pancreatic surgical resection. The purpose was to correlate DCE-MR quantitative parameters, (reflecting perfusion and/or permeability and the distribution volume fraction) with histologic features such as the degree of fibrosis and the microvascular density (MVD) in the corresponding tissues. A significant correlation was found between DCE-MR and histologic parameters: Ktrans was negatively correlated with the degree of fibrosis (high fibrosis was correlated with low perfusion), while the distribution volume fraction was positively correlated with the degree of fibrosis and with MVD (larger EES was correlated with high fibrosis and higher MVD). <p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Diabetes mellitus is amongst the leading causes of morbidity and mortality in the world, affecting young, adult and old people. Beta cell replacement therapy for insulin delivery remains the ultimate remedy for diabetes. However, insufficient donor pancreas and the use of immunosuppressive drugs prevent the wide-spread of this therapy. Other avenues of self generated beta cells within the organ itself need to be explored. Therefore, understanding the chronobiology of cellular mechanisms in the lineage of beta cell induced neogenesis is a valuable tool in improving beta cell replacement in patients with diabetes. The aim of this study was to induce recapitulation of the morpho-genetic sequence of endocrine cells development in the pancreas of rats after the pancreatic duct ligation (PDL) procedure. Serial sections of PDL tissues of the pancreas were obtained from 78 Sprague- Dawley rats and were assessed morphologically. The immunofluorescent tissues were statistically analysed using a computerized morphometry technique. The protein expression indices of Caspase3, Insulin, Pdx1, Ngn3, NeuroD and Pax6 were quantified. The efficiency levels of coexpression of these homeodomain proteins separately with insulin were defined by the ratio of the mean value of insulin expression to the mean value of their respective protein expression. The morphological changes were characterized by the appearance of granulated acinar cells at 6 hours post-PDL and the proliferation of endocrine tissues from 84 hours through to 120 hours. The morpho-immunofluorescent evaluation showed the highest immunoreactivity of Caspase3 and Pdx1 at 6 hours, Ngn3 at 36 hours, Pax6 and insulin at 84 hours while NeuroD expression was at 120 hours. The immunohistofluorescent analysis showed that caspase3 and Pdx1 were the first to be expressed at 6 hours while the insulin and NeuroD expression appeared later at 84 hours and 120 hours, respectively. However, Pax6 expression was continuous across time periods post-PDL, while Ngn3 expression showed a peak at 36 hours. The efficiency (highest and earliest expression) of co-expression of all these homeodomain proteins with insulin was restricted between 12 hours and 24 hours. The optimal efficiency was at 12 hours by Ngn3 with insulin. A good efficiency was shown for Pdx1 with insulin, NeuroD with insulin and Pax6 with insulin at 12 hours and 24 hours, respectively. A low efficiency was observed for insulin and caspase3 co-expression at 24 hours. This study suggests that for transplantation, PDL tissues harvested at an early time post-PDL (between 12 and 24 hours) could yield a higher success rate; the study also provides evidence for a connection between morphological changes in the PDL pancreas and the protein synthesis necessary for the lineage of endocrine cell development. / AFRIKAANSE OPSOMMING: Diabetes Mellitus resorteer onder die vernaamste oorsake van morbiditeit en mortaliteit wêreldwyd, en tuister jongmense, volwassenes en bejaardes. Daar bestaan egter ‘n wêreldwye tekort aan skenkerorgane met immuun-onderdrukingsterapie as ondersteuningsbehandeling. Beta-sel vervangingsterapie, vir die voorsiening van insulien, bly daarom die voorkeur behandeling vir die siekte wat noodsaak dat die wetenskap kyk na alternatiewe behandelingsregimens wat meganismes rondom orgaanregenerasie insluit. Begrip van die chronobiologie van die sellulêre meganismes betrokke rondom beta-sel ontwikkeling mag waardevolle lig werp op die neogenese van beta-selle wat gevolglik daartoe mag lei dat beta-sel vervanging as ‘n moontlike behandelingsterapie oorweeg mag word vir pasiënte met suikersiekte. Die oogmerk van hierdie studie is om die rekapitulasie van die morfo-genetiese volgorde van die endokriene pankreas na afbinding van die pankreasbuis te bepaal. Pankreasbuis afbinding is op 78 Sprague-Dawley laboratorium rotte onder algemene narkose uitgevoer, die pankreas is na voorafbepaalde tydsvakke verwyder en in histologiese seriesnitte gesny. Snitte is immunositochemiese gekleur en morfometries assesseer. Die afskeidingsindeks vir selboodskappers vir Caspase3, Insulien, Pdx1, Ngn3, NeuroD en Pax6 is kwantifiseer. Die gelyktydige afskeiding van elk van bogenoemde boodskappers tesame met insulien is omskryf as ‘n verhouding tot mekaar en in terme van dié van insulien. Die morfologiese verandering in die weefsel bespeur is gekenmerk deur die verskyn van gegranuleerde asinêre selle ses (6) ure na buisafbinding en die proliferasie van endokriene weefsel vanaf vier-en-tagtig (84) ure deurlopend tot een-honderd-en-twintig (120) ure. Die morfo-immunofluoresserende evaluering toon dat Caspase3 en Pdx1 by 6 uur die hoogste is, die van Ngn3 by 36 ure, Pax6 en insulien by 84 ure en NeuroD by 120 ure. Verder toon die analise dat Caspase3 en Pdx1 rondom 6 ure hul verskyning gemaak het terwyl dié van insulien en NeuroD eers rondom 84 tot 120 uur verskyn het. Die verskyning van Pax6 het deurlopend regoor al die tydsduurtes verskyn en Ngn3 het rondom 36 uur sy hoogste vlak bereik. Die gelyktydige uitdrukking van homeodomein proteïene tesame met insulien het slegs tussen die tydperke van 12 en 24 ure plaasgevind. Die uitdrukking van Pdx1 met insulien, NeuroD met insulien en Pax6 met insulien het almal tussen 12 en 24 ure plaasgevind. Caspase3 tesame met insulien is slegs by die 24 uur tydsperiode bespeur. Vir die oorplant van pankreas weefsel wat aan buisafbinding onderwerp is suggereer hierdie studie dat die geskikste tyd vir die oes van endokriene weefsel liewer vroeër (12 to 24 ure) as later uitgevoer behoort te word. Verder wil dit voorkom of hierdie tydsperiode ook die hoogste seltelling lewer. Die studie lewer waardevolle inligting oor die verwantskap tussen die morfologiese veranderings wat na buisafbinding plaasvind en die proteïen sintese wat sel-opvolgontwikkeling bevorder.

胰腺癌是一種致死率極高的惡性疾病,在全世界所有的癌症中死亡率排列第八, 在美國排列第四。 很多因素造成了胰腺癌較差的預後,其中包括: 早期檢出率極低; 較少胰腺癌患者的腫瘤適宜手術切除;高轉移率;以及對傳統放療和化療具有較高抗性等。 因此,發展新的治療藥物迫在眉睫。 / 近年來, 植物藥以及從這些植物藥裡分離出的天然化合物, 單獨使用或者與傳統化療藥物合併使用時, 都顯示出對不同類型的癌症具有較好療效。植物藥毛萼香茶菜(唇形科)含有豐富的具有抗癌活性的二萜類化合物。其中毛萼乙素(EriB) 是一個擁有最好抗癌活性的對映-貝殼杉烷型二萜化合物。 基於此背景, 本研究的目標為:利用胰腺癌體外體內模型, 研究EriB的抗胰腺癌活性以及誘導胰腺癌細胞凋亡的機理。 / 體外實驗中, EriB對四種胰腺癌細胞株都顯示了顯著的細胞毒活性,其活性與化療藥物喜樹堿類似。其中, EriB對胰腺癌細胞株CAPAN-2活性最強, 半數致死濃度IC₅₀為0.73 μM。細胞凋亡特徵:細胞核凝聚, 磷脂醯絲氨酸外翻, DNA梯狀條帶以及片斷化,在EriB誘導的胰腺癌細胞株CAPAN-2中出現。此外, EriB還造成癌細胞在細胞週期G2/M期的阻滯。機理研究發現, EriB是通過啟動絲裂原活化蛋白激酶(MAPK), caspase及 p53信號通路來誘導細胞凋亡和細胞週期阻滯的。抗凋亡蛋白與促凋亡蛋白比率(bcl-2/bak)的減少也可能對啟動細胞凋亡內途徑發揮一定作用。除此以外, EriB對癌細胞的細胞毒活性及致凋亡作用依賴于活性氧分子(ROS)的產生。在對細胞進行抗氧化劑預處理的實驗中發現, 只有含巰基基團的抗氧化劑能夠有效的阻斷EriB對癌細胞的活性。進一步實驗證明, EriB對細胞內兩個抗氧化系統: 谷胱甘肽系統及硫氧還蛋白系統的抑制作用導致了ROS在癌細胞中的積聚。同時,ROS的產生啟動了MAPK,熱休克蛋白70以及caspase信號通路,卻抑制了NFκB通路。 / 動物體內實驗證實, 每天對胰腺癌細胞移植瘤裸鼠進行腹腔注射EriB(2.5 毫克/千克),能有效的抑制腫瘤生長, 並且對心臟,肝臟和腎臟沒有引起顯著毒性。 對腫瘤組織的分析表明, 給藥組(EriB)比溶劑對照組出現更多的細胞凋亡, 並產生較多的ROS積聚。 / 綜上所述, 本項研究首次闡述了EriB具有顯著的體內外抗胰腺癌活性。機理研究證明, EriB抑制胰腺癌細胞內兩個含巰基基團的抗氧化系統, 從而導致ROS在細胞中積聚, 並啟動(或抑制)了包括MAPK, p53, caspase和NFB在內的信號通路, 最終導致癌細胞死亡。 此外, 動物體內研究證明EriB的抗腫瘤生長活性和低毒性, 令該化合物具有潛力進一步研究發展成為抗胰腺癌的新藥物。 / Pancreatic cancer is the fourth and eighth leading cause of cancer-related deaths in the U.S. and worldwide, respectively. Its poor prognosis is attributed to its late diagnosis, limitation to surgical resection, aggressive local invasion, and early metastases, as well as high resistance to chemotherapy and radiotherapy. Therefore, a search for an alternative to therapeutic agents is in desperate need. / In recent years, herbal medicines or natural compounds isolated from herbs either used alone or in combination with conventional anti-cancer agents have been shown to have beneficial effects on various cancers. In this context, the Chinese herb Isodon eriocalyx (Dunn.) Hara (family Lamiaceae) is a well-known source of anti-cancer diterpenoids, the most potent one being Eriocalyxin B (EriB, an ent-kauranoid). Therefore, the aims of the present study are to investigate the anti-tumor activities of EriB in human pancreatic adenocarcinoma cells and tumor-bearing mouse model, as well as the underlying mechanisms. / Our results showed that EriB exhibited significant cytotoxic effects on four pancreatic adenocarcinoma cell lines, with potencies being comparable to that of chemotherapeutic agent camptothecin. EriB had the most potent cytotoxicity in CAPAN-2 cells with IC₅₀ = 0.73 μM. The hallmark features of apoptosis, such as nuclear condensation, translocation of phosphatidylserine, DNA laddering, and DNA fragmentation were observed in EriB-treated CAPAN-2 cells. On the other hand, EriB also induced G2/M phase cell cycle arrest. Mechanistic studies revealed that EriB induced apoptosis and cell cycle arrest through the activation of MAPKs (p38, ERK1/2), caspase cascade, and p53/p21/cdk1-cyclinB1 signaling pathways. A decrease in the ratio of anti-apoptotic to pro-apoptotic proteins (bcl-2/bak) also contributed to the activation of intrinsic apoptotic pathway. Further investigation showed that EriB-induced cytotoxic and apoptotic effects were dependent on reactive oxygen species (ROS) production. Such demonstrated effects could be inhibited by pre-treatment with thiol-containing antioxidants. Furthermore, EriB induced ROS was mediated via the inhibition of two main antioxidant systems, namely glutathione and thioredoxin systems. EriB-mediated ROS activated multiple targets or signal pathways, including MAPK, heat shock protein (Hsp) 70, and caspase cascade, while inhibiting the NFκB pathway. / On the other hand, in vivo study demonstrated that daily intraperitoneal administration of EriB (2.5mg/kg/day) in human pancreatic tumor xenografts BALB/c nude mice significantly inhibited tumor growth, but without having toxicity in the heart, liver and kidney. In addition, EriB treatments induced in vivo cell apoptosis and superoxide production as observed in tumor tissues. / In conclusion, the present study reports for the first time that EriB has possessed anti-proliferative activities in pancreatic cancer cells. The anti-proliferative effects of EriB on CAPAN-2 cells could be attributable to the regulation of cellular apoptosis and cell cycle arrest. The inhibitory effects of EriB on two antioxidant systems result in the accumulation of ROS, which in turn activate MAPK, p53, Hsp70 and caspase cascade, while inhibiting NFB pathway and finally leading to pancreatic cancer cell death. Meanwhile, in vivo study further confirms the anti-tumor properties of EriB, suggesting that EriB could be considered as a potential chemotherapeutic agent for patients with pancreatic cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 207-230). / Abstracts also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Publications --- p.vi / Acknowledgements --- p.vii / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter Chapter1 --- General Introduction --- p.1 / Chapter 1.1 --- The pancreas --- p.2 / Chapter 1.1.1 --- Anatomy of the pancreas --- p.2 / Chapter 1.1.2 --- Histology of the pancreas --- p.4 / Chapter 1.1.3 --- Exocrine pancreas --- p.5 / Chapter 1.1.3.1 --- Structure of secretory acini, ducts and stroma in pancreas --- p.5 / Chapter 1.1.3.2 --- Functions of exocrine pancreas --- p.6 / Chapter 1.1.4 --- Endocrine pancreas --- p.9 / Chapter 1.1.4.1 --- Structure of islets cells --- p.10 / Chapter 1.1.4.2 --- Functions of endocrine pancreas --- p.10 / Chapter 1.1.5 --- Disorders of the pancreas --- p.11 / Chapter 1.2 --- Pancreatic cancer --- p.14 / Chapter 1.2.1 --- Epidemiology --- p.14 / Chapter 1.2.2 --- The risks and causes of pancreatic cancer --- p.15 / Chapter 1.2.3 --- Signs and symptoms of pancreatic cancer --- p.18 / Chapter 1.2.4 --- Types of pancreatic cancer --- p.19 / Chapter 1.2.5 --- Diagnosis of pancreatic cancer --- p.21 / Chapter 1.2.6 --- Staging of pancreatic cancer --- p.27 / Chapter 1.3 --- Treatments for pancreatic cancer --- p.29 / Chapter 1.3.1 --- Surgery --- p.29 / Chapter 1.3.2 --- Chemotherapy --- p.30 / Chapter 1.3.2.1 --- 5-fluorouracil (5-FU) --- p.32 / Chapter 1.3.2.2 --- Gemcitabine (Gem) --- p.33 / Chapter 1.3.2.3 --- Other cytotoxic agents --- p.34 / Chapter 1.3.3 --- Radiotherapy --- p.35 / Chapter 1.3.4 --- Target therapies --- p.37 / Chapter 1.3.4.1 --- Antiangiogenic therapy --- p.37 / Chapter 1.3.4.2 --- Epidermal growth factor receptor (EGFR) signaling inhibitors --- p.39 / Chapter 1.3.4.3 --- Hedgehog and Notch signaling pathways inhibitors --- p.41 / Chapter 1.3.5 --- Gene therapy --- p.42 / Chapter 1.3.6 --- Immunotherapy --- p.45 / Chapter 1.3.7 --- Combination therapies --- p.46 / Chapter 1.4 --- Molecular targets for pancreatic cancer chemotherapy --- p.49 / Chapter 1.4.1 --- Therapies-induced apoptosis --- p.49 / Chapter 1.4.1.1 --- Caspase cascade and bcl-2 Family --- p.49 / Chapter 1.4.1.2 --- Role of mitogen-activated protein kinases (MAPKs) in apoptosis --- p.50 / Chapter 1.4.2 --- Nuclear factor-κB activation in pancreatic cancer --- p.50 / Chapter 1.4.3 --- The PI3K and AKT pathway --- p.51 / Chapter 1.4.4 --- JAK/STAT pathway --- p.51 / Chapter 1.4.5 --- Other molecular targets --- p.52 / Chapter 1.5 --- Herbal medicine as an alternative treatment for cancer treatment --- p.53 / Chapter 1.5.1 --- Herbal medicines for different types of cancer treatment --- p.53 / Chapter 1.5.2 --- Herbal medicines for pancreatic cancer treatment --- p.59 / Chapter 1.6 --- Introduction of Isodon eriocalyx (Dunn.) Hara --- p.61 / Chapter 1.6.1 --- Background of Isodon genus and Isodon eriocalyx (Dunn.) Hara --- p.61 / Chapter 1.6.2 --- Diterpenoids from Isodon species and their activities --- p.62 / Chapter 1.6.3 --- The potential anti-cancer activity of Eriocalyxin B, a diterpenoid isolated from Isodon eriocalyx (Dunn.) Hara --- p.62 / Chapter 1.7 --- Aims and objectives of this study --- p.66 / Chapter Chapter 2 --- Eriocalyxin B induces apoptosis and cell cycle arrest in pancreatic adenocarcinoma cells through caspase- and p53-dependent pathways --- p.67 / Chapter 2.1 --- Introduction --- p.68 / Chapter 2.2 --- Materials and methods --- p.71 / Chapter 2.2.1 --- Preparation and quality control of Eriocalyxin B --- p.71 / Chapter 2.2.2 --- Materials --- p.72 / Chapter 2.2.3 --- Cell culture --- p.72 / Chapter 2.2.4 --- Preparation of human peripheral blood mononuclear cells (PBMC) --- p.73 / Chapter 2.2.5 --- Cytotoxicity assay --- p.75 / Chapter 2.2.6 --- Hoechst 33258 staining for morphological evaluation --- p.76 / Chapter 2.2.7 --- DNA fragmentation detection by DNA ladder --- p.76 / Chapter 2.2.8 --- Cell death detection ELISA --- p.77 / Chapter 2.2.9 --- Apoptosis detection by flow cytometry --- p.78 / Chapter 2.2.10 --- Cell cycle analysis by flow cytometry --- p.78 / Chapter 2.2.11 --- Western blot analysis --- p.79 / Chapter 2.2.12 --- Statistical analysis --- p.80 / Chapter 2.3 --- Results --- p.81 / Chapter 2.3.1 --- EriB induces cytotoxic effect in human pancreatic cancer cells --- p.81 / Chapter 2.3.2 --- EriB induces apoptosis in CAPAN-2 cells --- p.85 / Chapter 2.3.3 --- Activation of pro-apoptotic caspases in EriB-treated CAPAN-2 cells --- p.89 / Chapter 2.3.4 --- Modulation of bcl-2/bak ratio in EriB-treated CAPAN-2 cells --- p.92 / Chapter 2.3.5 --- EriB causes G2/M cell cycle arrest --- p.94 / Chapter 2.3.6 --- EriB modulates expression of G2/M cell cycle regulatory proteins through activation of the p53 pathway --- p.96 / Chapter 2.4 --- Discussion --- p.99 / Chapter Chapter 3 --- Eriocalyxin B induces apoptosis in pancreatic cancer CAPAN-2 cells via mediation of reactive oxygen species --- p.107 / Chapter 3.1 --- Introduction --- p.108 / Chapter 3.2 --- Materials and methods --- p.113 / Chapter 3.2.1 --- Materials --- p.113 / Chapter 3.2.2 --- Cell culture and MTT assay --- p.113 / Chapter 3.2.3 --- Apoptosis detection by flow cytometry --- p.114 / Chapter 3.2.4 --- Reactive oxygen species (ROS) detection by flow cytometry --- p.114 / Chapter 3.2.5 --- Glutathione assessment --- p.115 / Chapter 3.2.6 --- Glutathione peroxidase (GPx) activity detection --- p.116 / Chapter 3.2.7 --- Thioredoxin reductase (TrxR) activity detection --- p.116 / Chapter 3.2.8 --- Nuclear and cytosolic fractionation --- p.117 / Chapter 3.2.9 --- Western blot analysis --- p.117 / Chapter 3.2.10 --- Electrophoretic mobility shift assay --- p.119 / Chapter 3.2.11 --- Statistical analysis --- p.119 / Chapter 3.3 --- Results --- p.120 / Chapter 3.3.1 --- Thiol-containing antioxidants inhibits EriB-induced cytotoxic effects --- p.120 / Chapter 3.3.2 --- Thiol-containing antioxidants inhibits EriB-induced apoptotic effects --- p.122 / Chapter 3.3.3 --- Effects of EriB on hydrogen peroxide production --- p.125 / Chapter 3.3.4 --- EriB depletes glutathione level and suppresses GPx activity --- p.128 / Chapter 3.3.5 --- EriB inhibits thioredoxin system and activates ASK1 --- p.130 / Chapter 3.3.6 --- EriB increases Hsp70 and cleaved-PARP expression through ROS --- p.134 / Chapter 3.3.7 --- EriB inhibits NFkB pathway in CAPAN-2 cells --- p.137 / Chapter 3.4 --- Discussion --- p.142 / Chapter Chapter 4 --- In vivo study of the anti-tumor efficacy of Eriocalyxin B in human pancreatic tumor xenograft model --- p.149 / Chapter 4.1 --- Introduction --- p.150 / Chapter 4.2 --- Materials and methods --- p.154 / Chapter 4.2.1 --- Establishment of a subcutaneous pancreatic cancer xenograft model --- p.154 / Chapter 4.2.2 --- Evaluation of the effects of EriB on tumor growth --- p.155 / Chapter 4.2.2.1 --- Pilot study for EriB and camptothecin treatment --- p.155 / Chapter 4.2.2.2 --- Confirmation study of effective dose of EriB --- p.156 / Chapter 4.2.2.3 --- Dose-comparison study of CPT-11 --- p.156 / Chapter 4.2.2.4 --- Comparison study of EriB and CPT-11 treatments --- p..157 / Chapter 4.2.3 --- Measurement of plasma-specific enzyme levels --- p.157 / Chapter 4.2.4 --- Assays of terminal deoxytransferase-catalyzed DNA nick-end labeling (TUNEL) --- p..158 / Chapter 4.2.5 --- Histological evaluation --- p.159 / Chapter 4.2.6 --- Detection of superoxide by DHE staining --- p.159 / Chapter 4.2.7 --- Establishment of an orthotopic model (SW1990) of pancreatic cancer and detection of the plasma biomarker CA19-9 --- p.160 / Chapter 4.2.7.1 --- Detection of CA19-9 expression by immunofluorescent staining and western blot --- p.161 / Chapter 4.2.7.2 --- Establishment of an orthotopic pancreatic cancer xenograft model by SW1990 cells --- p.162 / Chapter 4.2.8 --- Statistical analysis --- p.164 / Chapter 4.3 --- Results --- p.165 / Chapter 4.3.1 --- EriB inhibits the growth of CAPAN-2 human pancreatic tumor xenografts --- p.165 / Chapter 4.3.2 --- EriB treatments induce cell apoptosis in tumor tissues --- p.173 / Chapter 4.3.3 --- Toxicity tests for EriB --- p.175 / Chapter 4.3.3.1 --- Plasma enzyme levels after EriB treatments --- p.175 / Chapter 4.3.3.2 --- No apparent alterations in histology of the heart, liver and kidney tissues --- p..176 / Chapter 4.3.4tEriB induces superoxide production in the tumor tissues --- p.178 / Chapter 4.3.5 --- Successful establishment of an orthotopic xenograft model --- p.180 / Chapter 4.4 --- Discussion --- p.184 / Chapter Chapter 5 --- General Discussion --- p.188 / Chapter 5.1 --- Discussion --- p.189 / Chapter 5.2 --- Conclusion --- p.204 / Chapter 5.3 --- Limitations of the study --- p.205 / Chapter 5.4 --- Future work --- p.206 / Chapter Chapter 6 --- References --- p.207

背景和目的：胰腺導管腺癌（簡稱胰腺癌）是世界範圍內惡性程度最高的癌癥之一，目前它的5 年生存率不到5%。大部分的病人在診斷初期就已經發展到了局部浸潤或遠處轉移的階段，因此失去了根治性手術切除的机会。輔助性化療對於胰腺癌病人來說是一個首選的治療方案，但是目前只有一小部分病人對化療藥物有良好的反應，而臨床化療失敗常與腫瘤細胞對化療藥物產生耐藥有關。吉西他濱是目前臨床上常用的一線抗癌藥物，但是它的耐藥現象在胰腺癌病人中廣泛存在，也是阻礙其臨床應用的主要原因之一。盡管已經有很多研究致力於揭示吉西他濱在胰腺癌細胞中的耐藥機理，目前臨床上仍然沒有有效的方法應對吉西他濱耐藥。我們的研究主要是為了探討一些以前沒有报道過的參與吉西他濱耐藥機理的基因，借此揭示胰腺癌細胞的吉西他濱耐藥的深層機制，為臨床上的治療提供理論依據。 / 實驗方法：我們實驗室之前在胰腺癌細胞株Capan2 中用全基因組RNAi篩選的方法確定LLGL1 作為抑癌基因能增強吉西他濱在胰腺癌細胞中的細胞毒性。我們隨後用體外細胞毒性分析實驗和皮下腫瘤動物模型來驗證LLGL1 是否能增強吉西他濱的細胞毒性，用蘇木素-伊紅染色和原味末端轉移酶標記技術分析抑制LLGL1 的表達是否會影響吉西他濱誘導的細胞雕亡反應。我們還應用微陣列分析技術進一步探尋LLGL1 的下遊靶蛋白，用實時定量PCR(qRT-PCR) 、蛋白印跡法（western blotting）、熒光素酶檢測等技術來進一步證實LLGL1 與下遊靶蛋白的關系，用免疫組織化學方法探究LLGL1 下遊靶蛋白在胰腺癌組織中的表達情況，以及該蛋白與LLGL1 的表達相關性，還應用染色體免疫共沈澱的方法探討轉錄因子Sp1(pThr453) 和RNA 聚合酶 II 在LLGL1 下遊靶蛋白的啟動子上的富集情況。 / 實驗結果：LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因的表達能誘導胰腺癌細胞對吉西他濱的耐藥，而上調該基因的表達則會增強胰腺癌細胞對吉西他濱的細胞毒性反應。OSMR 是LLGL1 的下遊靶蛋白, 其在胰腺癌組織中的表達與LLGL1 呈負性相關，抑制OSMR 的表達可以逆轉由LLGL1表達下調引起的吉西他濱耐藥現象。OSMR 表達上調可以增強腫瘤幹細胞標記物CD44 和CD24 的表達。另外，在胰腺癌細胞中，抑制LLGL1 的表達能激活ERK2/Sp1 信號通路，導致磷酸化Sp1(pThr453)的表達升高。OSMR 啟動子既沒有TATA 元件也沒有INR 元件，但是有Sp1 结合元件可供Sp1 結合。磷酸化Sp1(pThr453)可以結合到OSMR 啟動子的Sp1 结合元件上，從而促使RNA 轉錄酶II 結合到該啟動子上，啟動OSMR 基因的轉錄。 / 結論：我們的研究發現：1，LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因在胰腺癌細胞中的表達能上調OSMR 的表達，並誘導吉西他濱耐藥；2，OSMR 的表達在胰腺癌組織中與LLGL1 呈負性相關；3，下調LLGL1的表達能激活ERK2/Sp1 信號通路，進一步導致磷酸化Sp1(pThr453)和RNA 轉錄酶II 在OSMR 啟動子上的聚集，最終促使OSMR 的高表達，而下調LLGL1的表達能抑制該調節通路，從而抑制OSMR 的轉錄。 / Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Its 5-year survival rate is less than 5%, because most patients have already developed to the advanced stage of local invasion or distant metastasis once diagnosed, and missed the chances of curable surgical resection. Adjuvant chemotherapy is an alternative therapeutic strategy against PDAC. Yet, only very small proportion of patients could benefit from chemotherapy due to the innate and easily-acquired chemo-resistance in PDAC cells, especially to the first-line chemotherapeutic drug, gemcitabine. Many studies have been conducted to exploring the mechanisms underlying gemcitabine resistance in PDAC cells, but gemcitabine resistance is still the major obstacle impeding PDAC patients benefits from chemotherapy. Our studies aimed to investigate novel genes involved in gemcitabine response and to explore the undefined mechanisms generating gemcitabine resistance in PDAC cells. / Methods: Our colleagues previously performed genome-wide RNAi screening in gemcitabine-sensitive Capan2 cells. Lethal giant larvae homolog 1 (LLGL1) was identified as a potential gemcitabine-sensitizing gene which was then validated by our subsequent in-vitro drug cytotoxicity assay in LLGL1-inhibited Capan2 and SW1990 cells and in vivo subcutaneous xenograft mouse model. Hematoxylin & Eosin staining and terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling were applied for the assessment of apoptotic effects induced by gemcitabine in subcutaneous xenografts. We did gene expression microarray analysis to explore the potential downstream targets of LLGL1. Western blotting, qRT-PCR, and luciferase assay were applied to validate the downstream target of LLGL1 that were figured out by microarray analysis. We also did immunohistochemical staining to investigate the expression levels and correlationship of LLGL1 and its downstream target in PDAC specimens. Chromatin immunoprecipitation was performed to explore the enrichment of the transcriptional factor Sp1(pThr453) and RNA polymerase II (Pol II) at the promoter of the downstream targets of LLGL1. / Results: LLGL1 was identified as a gemcitabine-sensitizing gene, whose inhibition remarkably reduced gemcitabine response in gemcitabine-sensitive Capan2 and SW1990 cells, and ectopic expression induced gemcitabine response in gemcitabine-resistant PANC1 cells. Oncostatin M receptor (OSMR) was identified as a downstream target of LLGL1, whose expression was negatively correlated with LLGL1, and knockdown of OSMR significantly reversed gemcitabine resistance induced by LLGL1 inhibition in Capan2 and SW1990 cells. Additionally, activation of OSMR signaling was associated with the elevated expression of cancer stem cell markers, CD44 and CD24, both of which had already been identified to contribute to gemcitabine resistance in PDAC cells. Moreover, OSMR up-regulation induced by LLGL1 inhibition in SW1990 cells depended on the activation of ERK2/Sp1 signaling and subsequent accumulation of Sp1(pThr453) and Pol II at the TATA-less, INR-less but Sp1-binding-site-rich promoter of OSMR, while ectopic expression of LLGL1 in PANC1 cells inactivated ERK2/Sp1 signaling and subsequently reduced the enrichment of Sp1(pThr453) and Pol II at OSMR promoter. / CONCLUSIONS: Our studies revealed the novel tumor suppressive role of LLGL1 as a gemcitabine-sensitizing gene in PDAC cells. Loss of LLGL1 resulted in the activation of ERK2/Sp1 signaling and up-regulation of OSMR expression, and ultimately desensitized gemcitabine response in PDAC cells. More importantly, ectopic expression of LLGL1 disrupted such regulatory axis and improved gemcitabine response. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhu, Yinxin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 154-183). / Abstracts also in Chinese.