Aurora Kinase A Improves Acinar Cell Survival And Regeneration In Experimental Pancreatitis Of Mice

Part 1.Test the expression level of Aurora Kinase A in pancreas tissue with focus on miceObjective:To test the expression level of Aurora Kinase A in normal pancreas and acute pancreatitis tissue on adult mice,and to further explore whether the expression level of Aurora A in mice pancreas is regulated by inflammation.Methods:We are using repeated intraperitoneally injections of supramaximal cerulein for eight times per day and inject for one day to induce acute pancreatitis on adult BAC mice,we then collect pancreas tissue from mice in different time points(day 1-7)accordingly.To compare them with no cerulein treat group(day 0),we performed q RT-PCR,western blot and immunohistochemistry to examine the expression of Aurora Kinase A in different pancreas tissue from different treatment mice groups.Results:The q RT-PCR result showed the baseline expression level of Aurora A in pancreas on normal adult BAC mice is relatively low,and it has an increasing trend throughout the process of cerulein-induced pancreatitis in mice pancreas tissue.The highest Aurora A expression level was observed around 4-5 days after given the first injection of cerulein on mice,and it reached about 25 times as compared with no treat group,then it started to decrease afterwards.The western blot and immunohistochemistry of paraffin sections also suggested the similar trend with the q RT-PCR results,we found the Aurora A protein has significantly higher expression level in pancreas tissue at acute pancreatitis stage rather than in normal pancreas.The expression of Aurora A protein in mice pancreas samples showed a biphasic change curve within 1 week after the first dose of cerulein stimulation,it reached its peak around day 3-5,and it was significantly higher as compared with on day 0-3 or day 6-7.Conclusions:We found out the Aurora Kinase A is expressed in both normal pancreas tissue and in pancreas tissue of pancreatitis on mice,and its expression level has an obvious time-effect on cerulein-induced acute pancreatitis mice model.As we observed,Aurora Kinase A expression level has an obvious bi-changing trend followed by different time points after pancreatitis stimulation,and it is significantly higher expressed in pancreatitis than in no cerulein treatment mice group.Part 2.To generate Aurora Kinase A specific KO in pancreatic acinar cell in mice and to observe its phenotypeObjective:We are using tamoxifen inducible Cre-Loxp recombination system trying to generate Aurora Kinase A gene specific knockout in mice pancreatic acinar cells(AUO/BAC mice),and we got homozygous AUO/BAC tool mice by using this system and its phenotype has been observed.Methods: To generate the AUO/BAC mice,the Cre-Loxp system has been used on mice,i.e.we used homozygous AUO tool mice(AURKAflox/flox)which carried floxed Aurora A allele to cross-breed with Cre ERT mice(BAC)which carried Elastase promoter driven expression specifically in pancreatic acinar cells.Tamoxifen was given by oral gavage to induce the recombination in mice pancreas,and we expect to get homozygous AUO/BAC tool mice(Ela-Cre ERT;AURKAf/f)which has Aurora Kinase A contionally KO in the pancreatic acinar cells.After given mice tamoxifen to induce gene recommbination,we used q RT-PCR and verified the genotype in new mice.We calculated and compared the pancreas weight/whole body weight ratio between BAC mice and AUO/BAC mice.We also performed and compared the HE staining and immunofluorescence staining of DAPI in normal pancreas tissue of BAC mice and AUO/BAC mice.From those experiments above,we got to know the phenotype changes of Aurora Kinase A specific KO in pancreatic acinar cell in mice.Results: We successfully generated homo AUO/BAC mice in which Aurora Kinase A will be conditionally KO in pancreatic acinar cells under recombination.The genotype PCR verified the right genotype as we expected.As discussed above,because Aurora Kinase A has a relatively very low expression baseline level as we tested in the last part,so that we collected the pancreas tissue at day 3 after the first cerulein injection which have a relatively higher expression of Aurora A and supposed to have a clear view of comparison.We found the expression of Aurora A in pancreas tissue was significantly decreased in AUO/BAC mice as compared with BAC mice.Results of H&E staining and DAPI nuclear staining showed the histology of normal pancreas between these 2 kinds of mice were similar,except the pancreatic acinar cells in AUO/BAC mice showed accumulation of giant nuclei,which is a typical characteristic of polyploid cells,as well as increasead population of polyploid cells as compared with BAC mice.Conclusions:Deletion of Aurora Kinase A in pancreatic acinar cells in mice can resulted in nucleus changes in cell morphology of pancreas,but it showed no obvious influence on growth or survival both in pancreas tissue and in mice.Part 3.Effect of Aurora Kinase A specific knockout in pancreatic acinar cells on pancreatitisObjective:In order to test whether the conditionally KO of Aurora Kinase A in the pancreatic acinar cells can have effects on pancreatitis.We used cerulein-induced AP and CP model on both AUO/BAC and BAC mice groups,and we tested and compared between these two groups as followed.Methods:We first recuited the right genotypes of mice from the last two parts,and then split them into experimental group and control group as age and sex matched.Mice were fed with tamoxifen for several days and then rest for a couple of days in order to induce the genomic recommbinaiton.We used repeated i.p.injections of cerulein.Acute pancreatitis(AP)was induced by repeated intraperitoneally injections of cerulein at 100ug/kg/hr for 10 hourly.The histology of pancreas was evaluated,the pancreas/body weight ratio was caculatd and serum amylase level was measured.We used q RT-PCR to analyze m RNA expression levels of cytokines such as IL-1b,IL-6 and CXCL-10,we used the CD11 b,Ki-67 and Caspase-3 IHC-P stainings to examine inflammatory cells infiltration,cell proliferation rate and cell necrosis.Chronic pancreatitis(CP)was induced by repeated induced AP(cerulein at 100ug/kg/hr for 6 hourly)three time a week for 3 weeks and chronic pancreatitis was evaluated 1 week after the last injection for CP.We also compared with the pancreas/body weight ratio and histology of pancreas among two CP groups,we evaluatd pancreatic inflammation and macrophage infiltration via using CD11 b and F4/80 IHC stainings.Sirius-Red staining was also used to examine the collagen of chronic changes in the pancreas.Results: Acute pancreatitis and chronic pancreatitis were succefully induced by repeated cerulein injections in these two genotypes of mice.H&E staining showed that the AURKA deletion AP group had more acinar cell necrosis with relatively higher inflammatory cell infiltration in the pancreas than in the control group,q RT-PCR found the expression levels of IL-1b,IL-6 and CXCL-10 were significally higher in AUO/BAC-AP mice,and we found more positive cells in IHC-P stainings of CD11 b and Caspase-3 in AUO/BAC-AP group mice than in BAC-AP group mice.But the expression level of proliferative marker Ki-67 is relatively lower in AUO/BAC-AP mice,and there’s no significally difference of weight ratio or serum amylase levels among two groups.In the CP model,both H&E and Sirus-Red staining showed Aurora Kinase A deletion caused more fatty replacement with more acinar cell loss and fibrosis,the decreased pancreas/body weight ratio showed severe pancreatic atrophy in the AURKA deletion CP group as compared to BAC mice.(*p<0.01).The CD11 b and F4/80 postive cells in pancreas were substantially increased in AUO/BAC-CP mice.Conclusions:This part of study confimed that the conditionally KO of Aurora Kinase A in pancreatic acinar cells can increase the inflammation reaction of mice pancreas,which includes inflammtroy cell infiltration and cell necrosis,suggesting Aurora Kinase A protects acinar cell death in the early phase of pancreatitis,and it continued to influence the recover process of pancreatitis and pancreatic fibrosis.Our data showed Aurora Kinase A has protective effects on pancreatitis by preventing acinar cell death and promoting cell regeneration,Aurora Kinase A deletion in pancreatic acinar cells can aggravate the cerulein-induced pancreatitis on mice model.