The Effect Of PRSS1^R122H Mutation Related Trypsin Activation On Acute Pancreatitis And Mechanism

Acute pancreatitis is an inflammatory disease of the pancreas that causes significant morbidity and mortality.It is reported that acute pancreatitis is related with alchol,biliary stone,crapulent diet and congenital diseases.Some pancreatic congenital diseases showed PRSS1 gene dot mutation and the mechanism is still unclear.In this study,we applied BAC technique to create transgenic mice that can steadly carry PRSS1^R122H gene.The genotype and phenotype of the ransgenic mice were comfirmed.We also established caerulein-induced pancreatitis mice models to explore the relationship among PRSS1^R122H122H mutation,trypsin activation,acinar cell apoptosis and ADM during acute pancreatitis development.Part ? Detection of genotype and phenotype of PRSS1^R122H transgenic miceObjective:To detect the genotype and phenotype of PRSS1^R122H transgenic mice and to measure PRSS1expression.Methods and materials:We applied BAC technique to create transgenic mice that can steadly carry PRSS1^R122H gene.Mice tail genotyping were used to detect the genotype.We also observed the behavior,weighed the mice,sectioned pancreatic tissue to do HE staining of different ages of PRSS1^R122H transgenic mice.Results:PRSS1^R122H genotype was confirmed by DNA sequencing and mice tail genotyping.Western blot and qRT-PCR showed that PRSS1^R122H protein and mRNA expressed in transgenic mice turned out to be much higher than that of C57BL/6J mice,which is about 50%of PRSS1 ^R122H protein or mRNA in hereditary pancreatitis patients.The behavior,weight change,pancreas histology showed no significant difference between different ages of PRSS1^R122H transgenic mice and C57BL/6J mice.Conclusions:PRSS1^R122H genotype was confirmed.Behavior and weight change between PRSS1^R122H transgenic mice and C57BL/6J mice showed no significant differences and no spontaneous pancreatitis were observed in PRSS1^R122H transgenic mice.Part ? The effect of PRSS1^R122H gene mutation on mice acute pancreatitis modelObjective:To explore the effect of PRSS1^R122H gene mutation on mice acute pancreatitis model and sensitivity to cerulein.Methods and materials:L-Arginine and cerulein induced aute pancreatitis model were set.We bserved the behavior,weighed the mice body and pancreas tissue,measured serum amylase,sectioned pancreatic tissue to do HE staining of PRSS1^R122H transgenic mice to evaluate pancreatitis severity.We also did IHC to measure F4/80 and CD11b expression level.Cerulein was diluted to gradient concentration?1.25ug/kg?2.5ug/kg?5ug/kg?7.5ug/kg?10ug/kg?12.5ug/kg?15.0 ug/kg?to explore the lowest concentration which could induce acute pancreatitis.Results:L-Arginine induced aute pancreatitis model showed no significant inflammation in both PRSS1^R122H transgenic mice and C57BL/6J mice.Cerulein induced aute pancreatitis model with PRSS1^R122H transgenic mice showed significant higher pancreas/body weight ratio,serum amylase and sectioned HE staining scores than C57BL/6J mice.IHC result showed much higher expression of F4/80 and CD11b in PRSS1^R122H transgenic AP group.PRSS1^R122H transgenic mice showed severe acute pancreatitis with 12.5ug/kg cerulein injection,which is much lower than C57BL/6J mice.Conclusions:PRSS1^R122H gene mutation aggravated cerulein induced acute pancreatitis and sensitized pancreas response to cerulein stimulation.Part ? The effect of PRSS1^R122H mutation related trypsin activation on acute pancreatitis and mechanismObjective:To explore the effect of PRSS1^R122H mutation related trypsin activation on acute pancreatitis and acinar cell apoptosis.Methods and materials:Cerulein was injected intraperitoneally at a dose of 50ug/kg hourly and was given six times in total.We did HE staining,western blot and TUNEL detection for pancreas tissue from mice with cerulein injection for 24 hours and 72 hours.Trypsin inhibitor was applied after cerulein injection.We observed behaviors,calculated pancreas/body weight ratio,tested serum amylase,HE staining and IHC to measure the pancreatitis severity.We also did TUNEL detection to assess acinar cell apoptosis.Results:PRSS1^R122H transgenic mice showed higher trypsin activity after cerulein injection.Mice treated with cerulein showed significant higher pancreas/body weight ratio,serum amylase and HE histopathology scores within 24 hours and showed lower pancreas/body weight ratio,higher C-Caspase3 and?-SMA within 72 hours.Pancreas/body weight ratio was higher and C-Caspase3 and?-SMA were lower in PRSS1^R122H transgenic mice with trypsin inhibitor intake than mice treated by cerulein for72 hours.Conclusions:Active trypsin,cell apoptosis,ADM and fibrosis showed up after cerulein injection within 72 hours and all of them were alleviated after trypsin inhibitor intake.Based on the experiments above,the final conclusions are as follows:1.PRSS1^R122H genotype was confirmed.Behavior and weight change between PRSS1^R122H transgenic mice and C57BL/6J mice showed no significant differences and no spontaneous pancreatitis were observed in PRSS1^R122H transgenic mice.2.PRSS1^R122H gene mutation aggravated cerulein induced acute pancreatitis and sensitized pancreas response to cerulein stimulation.3.Active trypsin,cell apoptosis,ADM and fibrosis showed up after cerulein injection within 72 hours and all of them were alleviated after trypsin inhibitor intake.