Establishment Of A Model For Chronic Pancreatitis Induced By DBTC And Mechanism Of The Pathogenesis Of Pancreatitis Fibrosis In SD Rat

Partâ… Establishment of CP Model in SD Rats Induced by DBTCObjectiveTo establish a model of CP in Sprague-Dawley rat induced by dibutyltin dichloride(DBTC),and give intervention to the model rats by two drugs,and observe the pathological changes,pancreatic fibrosis degree,changes of serum AMS,and the effects of inflammatory cells infiltrating into the CP tissues during the pathogenesis of CP.MethodsTo apply DBTC by the single tail vein injection with a dosage of 0.8mg/kg which dissolved in 80%ethanol to the experimental group rats,after two days they were randomly grouped into three,Group A,B and C.Rats in Group A(30 rats) were treated without any medicine;Rats in Group B(26 rats) were injected Pentoxifylline(PTX) 6mg/(kg.d) into the abdominal cavity once a day;Rats in Group C(37 rats) were injected trichostatin A(TSA) 1ml(1μg/ml) into the abdominal cavity per week. Rats in the control group were injected the same volume of 80%ethanol through vena caudalis.The rats of experimental group were sacrificed respectively at 14d,28d and 56d after induction of the model,and all the rats of control group were killed at 56d.The pancreas and some major organs including liver,lung and kidney were inspected and scored by macroscopy and microscopy,at the same time,we would assess the degree of pancreatic fibrosis by Van Gieson's collagen staining,and detect the levels of serum amylase by automatic biochemistry assay apparatus;and mast cells by toluidine blue staining.Macrophages,CD₄⁺ and CD₈⁺ T cells were stained by EnVision immunohistochemical method.Results(1) Macroscopy:In Group A,B and C,there were some interstitial tissue edema,increased pancreas envelope tension,focal necrosis, hemorrhage and conglutination with peri-tissues in the 14d,28d and 56d groups;part of pancreatic tissues had local hard tuberculation,but all these pathological changes in Group A were more obvious than those in Group B and C.In control group,no pathological changes of pancreas were found.In experimental group,some cases of rats had bleeding point, focal necrosis and abscess in their livers and lungs.(2) Microscopy and Assessment:In control group,the histologic structures of pancreas were normal.In experimental groups,the pathological evaluation,fibrosis degree and prevalence of CP were significantly higher in the 56d group than those in the 14d group(P＜0.05 or P＜0.01).No significant differences were found between the prevalences of CP in Group A,B and C,but in Group A the prevalence of middle and severe CP in the 28d and 56d group were significantly higher than those in Group B and C (P＜0.05).In experimental group,the changes of liver and lung were in according with general changes.(3) Serum AMS:The levels of serum AMS of all the experimental group rats were significantly higher than those in the control group rats(P＜0.01).But no significant differences were found between the serum AMS of all the experimental group rats(P＞0.05).(4) Inflammatory cell counts:In experimental groups,the counts of macrophages and mast cells were significantly higher in the 56d group than those in the 14d(P＜0.05) and control group(P＜0.01).In Group B and C,the counts of CD₄⁺ were significantly lower in the 56d group than those in the 14d group(P＜0.01),while the counts of CD₈⁺ were on the contrary(P＜0.01).No significant differences were found between the CD₄⁺/CD₈⁺ratio of all the experimental group rats,but the CD₄⁺/CD₈⁺ ratio of all the experimental group rats was significantly lower than that of control group(P＜0.05 or P＜0.01)(5) Correlation analysis:In experimental groups,there were closely positive correlations among the pathological evaluation,fibrosis degree,macrophage and mast cell counts (P＜0.01).In Group A and C,negatively correlations were found between the pathological evaluations and CD₄⁺cell counts(P＜0.05 or P＜0.01).In Group A,there were closely negatively correlations between fibrosis degree and CD₈⁺cell counts(P＜0.01)Conclusion(1) The single tail vein injection of DBTC successfully induced CP in rats model.The method is characterized by simple interventional procedure,spending in short-time,relatively high preva- lence and cheap cost.But to some extent,DBTC has certain non-fatal toxic side-effect to several major organs,such as liver and lung.(2) The model of rats induced by DBTC was similar with human's CP tissue in pathological characters and levels of serum AMS.(3) In DBTC-induced CP, infiltrating macrophages,mast cells and CD₈⁺cells might play an important role in the initiation and development in chronic pancreatitis.Partâ…¡Mechanisms of the Pathogenesis of Chronic Pancreatitis Fibrosis in RatsObjectiveTo detect the correlation between the pathogenesis and progression of CP in rats and inflammatory cells,PSC and cytokines,and to study the effects of PTX and TSA interfering CP in rats.MethodsThe rat pancreatic tissues in experimental group and control group were both made to paraffin imbedding sections after 4%formalin fixation,a-SMA and desmin were stained by EnVision immunohistochemical method(two steps).PDGF-BmRNA,TGF-β1mRNA and CTGFmRNA were stained by in situ hybridization.Results(1) The counts of inflammatory cells were showed in Part One.(2) The counts of a-PSC in different time points were significantly higher in all the experimental groups than those in control group(P＜0.01);The counts of a-PSC in Group A were significantly higher in the 28d group and 56d group than those in the 14d group(P＜0.05);The counts of a-PSC in Group B and C were significantly higher in the 56d group than those in the 14d group(P＜0.05);(3) The counts of s-PSC in different time point rats were significantly lower in all the experimental groups than those in control group(P＜0.05),but no significant differences were found in different time point rats between the three groups(P＞0.05).(4)The positive expression rates of CTGFmRNA and PDGF-BmRNA and their scores in the 28d and 56d groups were significantly higher in Group B and C than those in control group(P＜0.01);The positive expression rates and scores of TGF-β1mRNA in Group A were significantly higher in the 56d group than those in control group(P＜0.05 or P＜0.01);The positive expression rates of TGF-β1mRNA in Group A and B were significantly higher in the 56d group than those in the 14d group(P＜0.05).The positive expression rates and scores of CTGFmRNA and PDGF-BmRNA in Group A were significantly higher in the 28d and 56d group than those in the 14d group (P＜0.05 or P＜0.01).(5) No significant differences were found between the counts of a-PSC and s-PSC and the positive expression rates and scores of TGF-β1mRNA,CTGFmRNA and PDGF-BmRNA in experimental group(P＞0.05).(6) The counts of a-PSC and the positive expression scores and counts of TGF-β1mRNA and CTGFmRNA and PDGF-BmRNA were significantly higher in the slight and middle and severe CP groups than those in control group(P＜0.01).The counts of a-PSC and the positive expression scores of CTGFmRNA and PDGF-BmRNA and their counts were significantly higher in moderate and severe CP groups than those in slight CP group(P＜0.05 or P＜0.01). The counts of s-PSC were significantly lower in slight and moderate CP groups than those in normal tissues(P＜0.01).(7) In experimental groups,significant positive correlations were found among the counts of a-PSC and the positive expression scores of TGF-β1mRNA and CTGFmRNA and PDGF-BmRNA and the pathological degree and fibrosis degree(P＜0.05 or P＜0.01).Conclusion(1) a-PSC might attend in the fibrosis of CP,and PDGF-BmRNA, TGF-β1mRNA and CTGFmRNA could activate PSC,and they might play an important role in pancreatic fibrosis.(2) Mast cell and macrophage were significantly related to PSC activation.(3) The mechanism of CP was very complex,so the drugs,such as PTX and TSA,which inhibit only one point of the mechanism couldn't control the progression of CP.