Erbin Expression In EMT Process Of Pancreatic Stellate Cell (PSC)mediated By TGF-β Pathway

Section 1 Isolation,identification and culture of mousepancreatic stellate cell by outgrowth from pancreatic tissueObjective: To isolate mouse pancreatic stellate cell by outgrowth from pancreatic tissue explanted into culture dishes,and to investigate the difference between the chronic pancreatitis model group and control group.Methods: Intraperitoneal injection of caerulein in mouse induced chronic pancreatitis,and to observe the fibrosis changes in pancreas by HE staining.Pancreatic tissues in chronic pancreatitis model group and control group were collected,washed by fetal bovine serum,minced into 0.5-1mm3,and cultured in sterile culture flasks.To identify mouse pancreatic stellate cells,morphological observation,oil red O staining,and immunocytochemical/immunocytofluorescent staining with α - SMA /Desmin /CK19 /SOX9/ Chymotrypsin /Amylase antibody were performed.To investigate the expression ofα-SMA /Desmin in m RNA,realtime fluorescence quantitative PCR was conducted between quiescent and activated pancreatic stellate cells.Result:(1)HE staining: pancreatic fibrosis and pancreatic acinar-to-ductal metaplasia occurred in chronic pancreatitis model group.(2)morphological observation: outgrowth cells in the 4th day look like oval or star,and are abundant in lipid droplets around nuclear;however,passage cells have larger sizes with the third dimension disappeared and the loss of lipid droplets;cells in CP model are smaller in size compared with control group.(3)Oil red O staining: outgrowth cells in early phase were stained positive in the two groups,and negative in passage cells.(4)Immunocytochemical /immunocytofluorescent: α-SMA and Desmin were positive in the passage cells of two groups;CK19,SOX9,Chymotrypsin and Amylase were negative in outgrowth cells.(5)realtime fluorescence quantitative PCR: compared to quiescent cell,the expression ofα-SMA m RNA increased in both passage cells and chronic pancreatitis model.Conclusion: Isolation of pancreatic stellate cell by outgrowth from pancreatic tissue did work in both control group and chronic pancreatitis model group,and outgrowth cells in early phase were quiescent in both two groups.Section 2 Difference of Erbin and TGF-β1 in PSC betweenCP model and control groupObjective: To compare expressions of Erbin and TGF-β1 in PSC between CP model and control group.Methods: CP models were induced by intraperitoneal injection of cerulean,PSCs outgrow from pancreatic tissue,and expressions of Erbin and TGF-β1 were compared between CP model and control group by realtime fluorescence quantitative PCR and Western blot,respectively.Results:(1)Erbin in CP model is distinctly increased compared with control group in m RNA(6.36:1)and protein(6.79:1)levels,P<0.05;(2)the expression of TGF-β1 is also increased in CP models with 3.99 folds in m RNA and 1.43 folds in protein compared with control ones,P<0.05.Conclusion: Both Erbin and TGF-β1 are up-regulated in pancreatic fibrosis process,and Erbin might affect pancreatic fibrosis through TGF-β pathway.Section 3 Expression of Erbin in EMT process induced byTGF-β1Objective: To explore whether the process of PSC activation induced by TGF-β1 is similar to EMT changes,and to investigate expressions of Erbin and TGF-β pathway in PSC activation.Methods: PSC outgrew from tissue in the 4th day is stimulated by 10ng/ml TGF-β1 for 48 hours,morphological characteristic is observed,and then the expressions of m RNA and protein are detected in both Erbin and moleculars of TGF-β/Smad pathway and ERK pathway by realtime fluorescence quantitative PCR and Western blot,respectively.Results:(1)PSC induced by TGF-β1 turn to a larger size with the third dimension disappeared and a loss of lipid droplets.(2)In PSC stimulated by TGF-β1,E-cadherin(0.33:1)is low expression,and α-SMA(2.91:1)is high expression in transcriptional level,P<0.05.Similarly,E-cadherin(0.07:1)is low expression,and α-SMA(3.40:1)is high expression in transcriptional level,P<0.05.(3)Erbin,TGF-β1,Smad 3 and ERK are all up-regulated in experiment group stimulated by TGF-β1 both in m RNA(13.03,2.94,2.08 and 2.99,respectively)and protein(2.28,1.30,3.15 and 1.73,respectively).Conclusion: The activated process of PSC is similar to EMT with upregulated expressions of Erbin,TGF-β/Smad pathway and ERK pathway,which is contributed to pancreatic fibrosis.