[in Japanese]

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Recent rapid progress in molecular biology has helped us understand the molecular pathogenesis of various diseases, including exocrine pancreatic diseases. However, our understanding has failed to enable the development of an effective therapy for pancreatitis or pancreatic cancer. Many agents aimed at specific key molecular targets often lead to disappointing results. There is considerable species variation in the physiology of the pancreas. Furthermore, we can make only a statistical inference on molecular defects that may be associated with pancreatitis or pancreatic cancer. In order to overcome these problems, we have to make a continuous effort to develop experimental models to test our hypotheses, which may lead to the development of treatments for pancreatic disorders.

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Experimental models for pancreatitis have been essential for research in acute and chronic pancreatitis because study of the early events in the development of human pancreatitis is almost impossible in both types of pancreatitis. Thus, a number of experimental models have been designed and modified to mimic clinical pancreatitis. Recently, it has become possible to prove the causal effect of a single protein or gene on the development of pancreatitis, using various strategies including molecular biology and genetic approaches. This review summarizes recent trends in the research for acute and chronic pancreatitis.

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Auto (self)-digestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. Although lysosomal cathepsin B is suggested to play a key role in intrapancreatic trypsinogen activation, it is not clear how trypsinogen is delivered to the lysosome. Autophagy is an intracellular bulk degradation system in which cytoplasmic components are directed to the lysosome/vacuole by a membrane-mediated process. To analyze whether autophagy delivers trypsinogen to the lysosome and whether this is related to the onset of acute pancreatitis, we produced a conditional knockout mouse lacking Atg5 (autophagy related) in pancreatic acinar cells. The severity of acute pancreatitis induced by cerulein was reduced in these mice. In addition, trypsin activation was greatly reduced in Atg5 deficient acinar cells. These data suggest that autophagy exerts a devastating effect in pancreatic acinar cells by activation of trypsinogen to trypsin. We propose that autophagy plays a major role in the pathogenesis of acute pancreatitis by delivering trypsinogen to the lysosome.

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HCO₃^- secretion by pancreatic duct depends on the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR anion channel, Na⁺-H⁺ exchanger (NHE3), SLC26A3 and A6 are localized in the apical membrane. In interlobular pancreatic ducts isolated from ΔF cystic fibrosis mice, cAMP-stimulated fluid secretion was abolished and apical Na⁺-H⁺ exchange activity was increased. The activated apical Na⁺-H⁺ exchange may acidify pancreatic juice in cystic fibrosis. In interlobular pancreatic ducts isolated from slc26a6 null mice, the exchange activity of luminal Cl^- and intracellular HCO₃^- was decreased, while the basal and cAMP-stimulated pancreatic HCO₃^- secretion in vivo was not affected. The role of SLC26 Cl^--HCO₃^- exchanger in HCO₃^- secretion should be examined in guinea-pig pancreatic ducts which are able to generate more HCO₃^--rich pancreatic juice.

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As an animal model of severe acute pancreatitis (SAP), rat bile salt-induced pancreatitis is frequently employed. However, in vivo models have limitations concerning the analysis of the molecular mechanism. We have already disclosed that apoptosis is involved in the mechanism of organ dysfunction during SAP, and that PAAF (pancreatitis-associated ascitic fluid) has the ability to induce apoptosis. Recently, we showed that the breakdown of the intestinal mucosa via accelerated apoptosis increased the intestinal permeability in rat SAP. To investigate the mechanism of increased permeability, we established an in vitro model. T84 cells were grown on Transwell, and PAAF was added to the basolateral side of the well. TEER (transepithelial electrical resistance) was measured for the assessment of cellular permeability. This model well reproduced the in vivo phenomenon, meaning that PAAF decreased TEER without affecting the tight junction and induced apoptosis in T84 cells. We think that this model will be useful for investigating the molecular mechanism of organ dysfunction and bacterial translocation during SAP.

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Lack of an appropriate animal model for chronic pancreatitis hampered investigations of the pathophysiology and appropriate therapy. We examined whether a disturbance of pancreatic juice (PJ) flow causes pancreatic fibrosis in rats. First, we infused a viscous liquid, agarose (0.01%; 40μl/100g body weight) into the pancreatic duct to disturb the PJ flow. Infusion of agarose solution together with low concentration (0.1%) sodium taurocholic acid caused marked fibrosis and inflammatory cell infiltration in the pancreas for 4 weeks. Next, we elevated the hydrostatic pressure in the pancreatic duct by raising the free end of the pancreatic cannula to the vertical position to cause a disturbance, but without interruption, of the PJ flow. Elevation of the hydrostatic pressure caused pancreatic fibrosis and inflammatory cell infiltration in the inter- and intralobular areas similar to human chronic pancreatitis after 2 weeks. In the present study, we have demonstrated two experimental models of chronic pancreatitis.

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Human pancreatic myofibroblats are useful for the analysis of cellular responses in the pancreas. Two types (human and rat) of pancreatic myofibroblasts have been reported. When compared with rat myofibroblasts, human pancreatic myofibroblasts have several beneficial points: e.g. (1) behavior of human cells may reflect in vivo human responses more precisely, (2) more experimental tools, such as antibodies, are available for human than rat, and (3) due to the complete analysis of human genes, molecular approaches are more easily applicable to human cells than rat cells. In this study, we analyzed IL-1β induced genes in human pancreatic myofibroblasts by means of DNA microarray. IL-1β induced many genes associated with inflammatory and immune responses, such as chemokines and interleukins. Among them, we are now investigating IL-32 expression in these cells. Since IL-32 has been reported as a TNF-α-inducing factor, IL-32 expression in human pancreatic myofibroblasts suggests that IL-32 may be involved in the pathophysiology of acute and chronic pancreatitis.

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To establish a pancreatic cancer model in mice, dimethylbenzanthracene (DMBA) was injected into mice pancreata. By 3 months after DMBA injection into the pancreas, 6 of 10 mice showed visually recognizable tumors with precursor lesions of various types of cell atypia. The expression profiles of smad 4, cyclin D1 and p53 in the DMBA-induced tumors were similar to those of human pancreatic cancer, suggesting that this would be a useful mouse model for studying the morphologic and molecular mechanisms involved in pancreatic carcinogenesis. Semi-quantitative reverse transcription-polymerase chain reaction with microdissection demonstrated that Notch1 expression was continuous from precursor lesions to carcinoma cells compared to precursor lesions. However, no mutations of K-ras gene were detected in precancerous or cancerous lesions. Therefore, a DMBA model may be useful for understanding the mechanism underlying the transition from precursor lesions to carcinoma since such a model can show this progression without K-ras activation.

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We present a hamster model for the induction of intraductal papillary carcinoma (IPC) in the main pancreatic duct that mimicks human intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Hamsters underwent cholecystoduodenostomy with dissection of the distal end of the common duct. In this hamster model, reflux of bile into the pancreatic ducts was recognized and IPCs were frequently induced in the main pancreatic duct after the administration of a chemical carcinogen. The induced IPCs showed many similarities with human IPMN, i.e., the tumor development from the main or large pancreatic ducts, intraductal papillary growth with a histological pattern of papillary proliferation of tumor cells, and low-grade malignancy. Our hamster model could be a useful animal model for investing human IPMN.

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We developed short-term pancreatic cancer models in hamsters using PGHAM-1 cells and estimated the utility of the models for research on metastasis and therapeutic trials. Using three PGHAM-1 models; 1) primary pancreatic cancer and simultaneous liver metastasis by intrapancreatic transplantation, 2) liver metastasis alone by intrasplenic transplantation, 3) peritoneal dissemination by intraperitoneal transplantation, within twenty-one days after inoculation, we studied the specific characteristics of metastases and the effect of several anti-angiogenic substances on primary and metastatic pancreatic tumors. Vascular endotherial growth factor and the anatomical character were important factors for metastasis. In the therapeutic experiment, the incidence, size, diameter, microvessel density (MVD), and apoptotic index of the tumors were preferably influenced by the anti-angiogenic substances. In addition, PGHAM-1-Luc, i.e., luciferase-positive PGHAM-1 cells, were newly developed. These models would be suitable not only for studying the pathogenesis and metastasis of pancreatic cancer but also for preclinical trials of chemotherapeutic agents such as anti-angiogenic substances.

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Pancreatic ischemia and arterial occlusion due to the formation of microthrombosis in the pancreatic vessels caused by the up-regulation of coagulability may have an important role in the development of mild pancreatitis into necrotizing pancreatitis. Recently, vasospasm has been noticed as a cause of exacerbation of acute pancreatitis. Angiography of abdominal vessels in the early phase of acute necrotizing pancreatitis showed ischemic changes with vasospasm in accordance with the poorly perfused area of the pancreas detected by CE-CT. Severe ischemic changes on angiography were primarily observed in patients in whom more than 50% of the pancreas was poorly perfused. The extent of the ischemic changes with vasospasm was correlated with the extent of the poorly perfused area of the pancreas and the mortality rate. Non-occlusive mesenteric ischemia is often accompanied by severe acute pancreatitis, and severe vasospasm occurred in both the celiac artery and the superior mesenteric artery and their branches simultaneously. These findings demonstrate that vasospasm is a major cause of pancreatic ischemia in acute necrotizing pancreatitis.

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A 50-year-old male, with a complaint of epigastric pain, visited a nearby clinic. CT demonstrated a mass in the pancreatic head, and he was referred to our hospital. Biochemistry showed elevated CA19-9, CEA, and amylase. Image modalities revealed diffusely swollen pancreas, a 30-mm mass at the pancreatic head with dilatation of the distal main pancreatic duct, and multiple 10-25-mm liver masses. Pancreatic juice cytology revealed cancer cells, and pancreatic cancer with liver metastasis complicated with pancreatitis was diagnosed. After the pancreatitis healed, gemcitabine (1,400mg, day 1, 8, and 15; off, day 22) plus tegaful/uracil compound (300mg, every day) were given. The tumor decreased in size until treatment termination due to interstitial pneumonia. With brain and skin metastases, he died on day 348. Although rare, brain and skin metastases of pancreatic cancer should be kept in mind as survival can be improved by advanced chemotherapy.

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A 73-year-old man with a long history of alcohol abuse was admitted to our hospital because of severe epigastric discomfort. He was diagnosed as an acute exacerbation of chronic pancreatitis based on findings such as calcification of the pancreas, dilation of the main pancreatic duct, and elevated serum amylase level. In the pancreatic head, a mold-shaped calculus measuring 22×12mm was incarcerated in the main pancreatic duct. Endoscopic papillosphincterotomy and extracorporeal shock-wave lithotripsy (ESWL) were performed. Eight sessions were necessary to crush the calculus by ESWL. However, small calculi still existed in the pancreatic duct. Although the guidewire was successfully advanced across the stricture, the catheter could not be advanced. To dilate the refractory stricture, the Soehendra stent retriever (SSR) (Wilson-Cook Medical, Inc.) was employed. As a result, the catheter was successfully advanced across the stricture. Finally a pancreatic stent was inserted into the pancreatic duct. The patient was then discharged and had no symptoms. The SSR is feasible for treating intractable pancreatic duct stricture.

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A 64-year-old man was admitted to our hospital because of upper abdominal pain. Abdominal computed tomography revealed chronic pancreatitis with pancreatic stones and choledocholithiasis. The endoscopic procedure was unsuccessful. We performed surgical transduodenal papillotomy and removal of an incarcerated stone of 15mm in diameter. The stone was a pancreatic stone and there were many other pancreatic stones in the dilated duct and parenchyma of the head and untinate process of the pancreas. We therefore performed coring out the head of the pancreas with side to side pancreaticojejunostomy (Freys procedure). The incarceration of pancreatic stones in biliary system is very rare, but we should consider the possibility of this situation in the diagnosis of obstructive jaundice in patients with pancreatolithiasis.

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