| BackgroundAcute pancreatitis is a common clinical acute abdomen.10~20% of the patients develop into severe acute pancreatitis(SAP)。The mortality rate of SAP is about 15~20%,and the prognosis is unfavorable . Especially, The mortality rate of the early severe acute pancreatitis(ESAP) combined with multi-organ dysfunction syndrome (MODS) is more higher. So, SAP is considered one of the unsolved problems of life science in 21st century. How to prevent the progressing aggravation of SAP, and promote quickly reparation of damaged pancreas tissue? In recent years, some authors have made large of penetrating studies for pathogenesis and corresponding therapy methods in pancreatitis, and they have get some outcomes. However, the pathological change can’t be explained perfectly and satisfactorily so far. The research on stem cell has gradually become a hot point in recent ten years. And so, more and more scholars pay close attention to the research on pancreatic stem cell which are involved in the repair of damaged pancreatic tissues..The stem cell is a kind of cell which can keep alive for a long term and has a few of differentiation potencies. While, the progenitor cell is a kind of cell keeping one or double ways of differentiation, and the capability of its differentiation, self-renew and self-maintenance is confined. For the survival time, the progenitor cell is shorter than the stem cell. So far, there are some problems which can not be solved in stem cell involved in pancreas injury reparation. (1)Is really there primary stem cell in adult pancreatic tissue? (2)Where is the stem cell in pancreatic tissue? (3)How does the stem cell involve in pancreas injury reparation? Some authors found that the primary pancreatic stem cell allocates in pancreatic islet, duct epithelium, and gland alveolus epithelium. However, some authors have showed their suspicious that there may be no primary pancreatic stem cell in adult pancreatic tissue, and they were sure that the pancreatic stem cell come from the marrow mesenchymal stem cell actually. Is really there primary pancreatic stem cell in adult pancreatic tissue? The collagen fiber inevitably proliferate and deposit in tissue when pancreas is damaged. In this situation, what a role does the stem cell have in the pathophysiologic process of acute pancreatitis(AP)? In this study, we will establish rat AP animal model, regulate the apoptosis of acinous cell, and observe the pathophysiologic process that the fiber and pancreatic stem cell are involved in pancreatic injury reparation.Objective In this study, we will establish rat AP animal model, and approach the contents as follow: (1) The natural pathophysiologic progress of AP. (2) The apoptosis rule of acinous cell and the influence of dexamethasone for apoptosis in the pathophysiologic process of AP. (3) Approaching the location of primary stem cell and migrating rule of stem cell in damaged pancreatic tissue with immunohistochemistry of specific antigen of pancreatic stem cell. (4) Approaching the generating rule of pancreatic stem cell with immunohistochemistry of Brdu, which is the specific proliferative antigen of cell. (5)Approaching the distribution rule of typeⅠcollagen in pancreatic injury tissue, and approaching the regulatory role of TGF-β1 for typeⅠcollagen with real-time PCR quantitative analysis for TGF-β1 mRNA, which is the important controlling gene of typeⅠcollagen. In this study, we will initially elucidate the location of primary stem cell in the pancreatic tissue, and then, elucidate the role of apoptosis of acinous cell and pancreatic stem cell in pancreatic injury reparation. And further, we could be develop a new way of therapy for AP from cytology.Methods1. Grouping and management methods Eighty four SD rats were randomly divided into experimental (30 rats), interventional (30 rats) and control groups (24 rats). Every group was randomly and averagely divided into 6 sub-groups too, and every sub-group has 5,5,and 4 rats respectively.(1) The experimental and interventional group were given intraperitoneal injections of cerulein to induce acute pancreatitis, 50ug/Kg, once a hour, and continuously 4 times. (2) The control group was given intraperitoneal injections of 0.5 ml normal sodium in the same time. (3) The interventional group were given intraperitoneal injections of dexamethasone, 0.5mg/kg, once a day. (4)At 6 h, 1 d, 2 d, 3 d, 5 d and 7 d following pancreatitis induction, one sub-group rats from every group were sacrificed. (5) Afterwards, 5-bromo-2’-deoxy-uracil nucleotides (Brdu) were intraperitoneally injected 6h and 3h prior to sacrifice in order to label the proliferative pancreatic cells, 100mg/kg once.2. Operational methodsThe rats were anesthetized by aether inhalation. When opened the abdominal, the organs and ascitic fluid were observed; 2~4ml blood was drew from apex of heart, which can be ready for amylase detection; 200μg tissue were obtained from middle of pancreas for RT-PCR, which was stored in liquid nitrogen; 500mg tissue were obtained from middle of pancreas for HE, apoptosis and immunohistochemistry, which was stored with aldehyde fixation.3. Inspection items and methods(1) Serum amylase with automatic biochemistry inspection system; (2) The pathological changes of pancreatic tissue were observed through conventional HE staining.; (3) The cell apoptosis of pancreatic tissue was inspected according to the directions of apoptosis kit; (4) The pancreatic stem cell marker Nestin, C-kit, and the cell proliferation marker Brdu, and then the typeⅠcollagen were detected with immunohistochemistry; (5)The expression level of an important pancreatic stem cell regulatory factor, pancreatic duodenal homeobox 1 (PDX-1), was determined by real-time PCR; (6) The expression level of an important typeⅠcollagen regulatory factor, TGF-β1 was determined by real-time PCR.Results1. The pathophysiological change The pancreatic tissue of control group display pink, soft, and no edema. Under a light microscope, the construction of pancreatic tissue was clear. The pathophysiological progression of experimental and interventional group is similar. Between 6h to 1d, there was small amounts of bright ascites in the abdominal cavity, and the specimens generally display a high degree of edema without obvious hemorrhage. Under a light microscope, acinar cells showed apparent edema with a lot of vacuole-like structures dispersed in the pancreatic acini and islets. The peak amount of vacuole-like structures occurred in 1d. After Day 1, the amount of vacuole-like structures gradually decreased. The interlobular space significantly increased, which was likely caused by inflammatory edema. A small number of inflammatory cells were found to be clustered in the interlobular region and the cells gradually spread between the acini. On 2~3d, the edema of the specimens was significantly diminished compared to the edema observed in the previous phase with dispersed hemorrhagic spots. Interlobular vascular damage was detected with a microscope along with an effusion of red blood cells from the vascular lumen. A large number of red blood cells accumulated in the interlobular region. Some of the pancreatic lobules exhibited patchy necrosis. Acinar-like cells were rarely observed in the necrotic regions, but large amounts of inflammatory cells were found in these areas. On 5 ~7d, pancreatic edema subsided and there were patches of dark red. The reduction in the number of red blood cells accumulated between the acini and in the interlobular region was observed under a microscope. A clear boundary was detected between the necrotic area and the normal acinar cells. Large quantities of inflammatory cells were found in the necrotic region. The interacinar space returned to normal and vacuole-like structures were rarely observed.2. The apoptosis analysisFew apoptosis cells could be seen in normal pancreatic tissue. The apoptosis progression of experimental and interventional group is similar. In damaged pancreatic tissue, the apoptosis cell nucleuses and apoptotic bodies were stained buffy; The cell envelopes of some apoptosis cells had splited, and mixed together, then displayed a big vacuole-like structures, in which some apoptotic bodies could be seen; In any time, the apoptosis cells could be seen in pathophysiological progression; The apoptosis index number (AI) went up quickly after 6h, and the peak amount of AI occurred in 1d. After 1d , AI gradually decrease. The AI of experimental and interventional group was obviously higher than control group in the same time(P<0.05).3. Serum amylase analysisThe quantity of Serum amylase in control group was 1200～1900 U/L; The tendency of Serum amylase in experimental and interventional group was alike; On 6h, the quantity of Serum amylase in experimental and interventional group went up sharply, and got the peak; From 1d, the Serum amylase decreased , and which got to normal in 7d. From 6h~3d, the quantity of Serum amylase in experimental and interventional group was more higher than the control group(P<0.05). However, there was no obviously difference in the 3 groups after Day 7 (P>0.05).4. The distribution characteristics of nestin-positive cellsThe nestin protein was expressed at a low level in normal pancreatic tissues. Six hours after the disease model prepared, the nestin-positive cells were mainly found in the lumen of interlobular vessels in clusters. A small number of positive cells were found around the vascular cavity. One to two day after the cerulein was injected, the nestin-positive cells were still primarily distributed in the interlobular region and in the vascular cavity surrounding the tissues. Some of these cells had an outside distribution along the vascular lumen in two parallel lines. On Day 3, large numbers of nestin-positive cells were found in the pancreatic lobules, and the positive cells in the pancreatic islets increased significantly. On 5~7d, the number of nestin-positive cells were significantly reduced within the pancreatic lobules and the islets. Few nestin-positive cells were observed in the interlobular region and within the lumen of the interlobular vessels.5. The distribution characteristics of C-kit positive cellsThere were few C-kit positive cells in normal pancreatic tissue; In pathophysiological progression(6h~7d), C-kit positive cells could only be found in pancreatic islets all the time, and the color was stained from light to thickness, and then light. The positive cells could not be seen out of islets all along.6. The distribution characteristics of Brdu-positive cellsFew Brdu-positive cells could be seen in normal pancreatic tissue; 6h~1d after the pancreatitis model prepared, there still were few Brdu-positive cells in tissue; On 2~3d, a small amounts of Brdu-positive cells could be found in the interlobular region; On 5d, there were large number of positive cells in glandular lobule, and too, amounts of Brdu-positive cells distributed in the region of few inflammatory cells, and some positive cells connected to sheets. On 7d, there were few positive cells in relatively normal pancreatic tissue, and larges of Brdu- positive cells occupied soundly in the region of few inflammatory cells.7. The distribution characteristics of typeⅠcollagen proteinFew positive protein could be found in normal pancreatic tissue. 6h after the pancreatitis model prepared, the positive stain firstly appeared in acinar cells around the glandular lobules, and which were stained lightly. From 1d to 7d, the position of positive stain kept in the same region. howevere, the color of positive cells gradually become thick, and positive cells gradually distributed to straps. The centre of glandular lobule showed lightly stained, and there were no positive stain in pancreatic islets all the time.8. The expression of PDX-1 mRNAPDX-1 mRNA was positively expressed in the normal pancreatic tissues, but at a low level. After pancreatitis was induced, the expression level of the PDX-1 gene gradually increased and reached its peak on Day 3. The expression gradually decreased afterwards and got close to a normal level on Day 7. The relative quality of expression of experimental group was more higher than which of control group in the same time(p<0.05).9. The expression of TGF-β1 mRNA TGF-β1 mRNA was positively expressed in the normal pancreatic tissues, but at a low level. After pancreatitis was induced, the expression level of the TGF-β1 gene gradually increased and reached its peak on 7d. The relative quality of expression of experimental group was more higher than which of control group in the same time(p<0.05).Concludes1. The apoptosis of acinar cells is a chain reaction like waterfall in pathophysiological progression of AP all the time, and which reaches a peak at the early stage of inflammation due to the self-protective mechanism of pancreatic tissues against self-digestion; The vacuole-like structures in early stage of inflammation are probably the remaining spaces after apoptosis of acinar cells; Dexamethasone can’t obviously promote apoptosis for acinar cells.2. Because of the very important role of apoptisis for acinar cells, The pathological changes of AP can be divided into three phases: the edema and apoptosis phase (6 h– 1 d), the hemorrhagic necrosis phase (2 d– 3 d) and the reconstruction phase (5 d– 7 d). which can offer more help to understand the pathophysiological progression of AP.3. Primary pancreatic stem cells may not exist in the adult pancreatic tissues. The so-called pancreatic stem cells, or nestin-positive cells, may actually originate from bone marrow mesenchyme stem cells.4. The C-kit positive cells are a kind of progenitor cells of pancreatic islets, which only are involved in the repair of damaged pancreatic islets, and have no migrating characteristic.5. PDX-1 mRNA plays an important regulative role in which the stem cells are involved in the repair of damaged pancreatic tissue.6. TypeⅠcollagen plays a important role involving in the repair of damaged pancreatic tissue, and TGF-β1 mRNA is the critical regulative gene of TypeⅠcollagen. |
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