| According to statistics,hypertriglyceridemic pancreatitis(HTGP)has become the third leading cause of AP in China in recent years,but its pathogenes is has still not been clarified.The toxic effect of FFAS in hypertriglyceridemia and the injury of acinar cells caused by intracellular calcium overload are currently recognized as the pathogenesis of HTGP.However,the above studies all focus on pancreatic acinar cells.Pancreatic ductal epithelial cells and their secreted mucus form pancreatic ductal mucosa barrier(PDMB).Moreover,pancreatic ductal epithelial cells extend to the acinar lumina and are contiguous to acinar cells.Our previous animal experiment demonstrated that myosin light-chain kinase(MLCK)and Cav1.2 expressions were up-regulated and tight junction(TJ)was changed in pancreatic tissue of an AP model of HTGP rats induced by intraperitoneal injection of caerulein.According to literature,obvious fatty change was not found in pancreatic acinar cells in the rat model of hypertriglyceridemia.We speculate that FFAS may directly cause damage to pancreatic duct epithelial cells and their constituent PDMB,resulting in the pathogenesis of HTGP.Whether MLCK,Cav1.2 and TJ are involved in the pathogenesis of HTGP is preliminarily studied in this study.Section I Effect of high fat on MLCK and intercellular TJ in pancreatic duct epithelial cellsObjective: To observe the effect of high fat on MLCK,TJ-related proteins and Ca2+ channel,by co-culturing oleic acid(OA),palmitic acid(PA)and HPDE6C7 cells to stimulate in vivo high fat environment.Methods: HPDE6C7 cells were treated with PA at different concentrations(100,200 300,400,500 ?M/L)and oleic acid at different concentrations(100,200 300,400,500?M/L)for 24 h,respectively.Cell proliferation was detected using CCK-8 assay.The mRNA expression of MLCK,ZO-1 Claudin-2,Occludin,Cav1.2,Cav1.3,Cav2.1,Cav2.2,Cav3.1,and Cav3.2 was detected by q PCR.Results: 1.(1)OA at 300 ?M,400 ?M and 500 ?M inhibited the proliferation of HPDE6 cells,which was the most obvious at 500 ?M(P < 0.05).(2)PA at 200 ?M,300 ?M,400 ?M and 500 ?M inhibited the proliferation of HPDE6 cells which was the most apparent at 500 ?M(P < 0.05).2.(1)OA at 400 ?M promoted the mRNA expression of MLCK(P<0.05),and oleic acid at 300 ?M inhibited the mRNA expression of Occludin(P<0.05).OA at different concentrations all inhibited the mRNA expression of ZO-1(P<0.05).OA showed no effect on the mRNA expression of Claudin-2(P>0.05).(2)PA promoted the mRNA expression of MLCK in concentration dependence(P<0.05).PA at 200 ?M inhibited the mRNA expression of Claudin-2 and Occludin(P<0.05),and PA at 100 ?M-300 ?M inhibited the mRNA expression of ZO-1.3.(1)OA at 400 and 500 ?M promoted the mRNA expression of Cav2.2,and OA at 300 ?M,400 ?M and 500 ?M promoted the mRNA expression of Cav1.2?Cav1.3?Cav2.1?Cav3.1 mRNA expressio(P<0.05).Different concentrations of OA had no effect on Cav3.2(P>0.05).(2)PA at 200 ?M and 500 ?M promoted the mRNA expression of Cav1.2 and Cav2.1.PA at 300 ?M,400 ?M and 500 ?M promoted the mRNA expression of,Cav2.2 and Cav3.2 and PA at 200 ?M-500 ?M promoted the mRNA expression of Cav1.3and Cav3.1.Conclusions: 1.OA and PA inhibit the proliferation of human pancreatic ductal epithelial cell line HPDE6C7,which was the most obvious at 500 ?M.2.OA and PA up-regulate the mRNA expression of MLCK,Cav1.2,Cav1.3,Cav2.1,Cav2.2 and Cav3.1 in HPDE6C7 cells,and down-regulate the mRNA expression of ZO-1,Occludin and Claudin-2.Section II Expression changes of MLCK in cerulein-induced HTGP pancreatic ductal epithelial cell modelObjective: To observe the effect on MLCK,TJ-related proteins and Ca2+ channel with CAE stimulating HPDE6C7 cells co-cultured with PA to stimulate the pathogenes is of HTGP in vivo,and then ML-7 intervention.Methods: PA at 200 ?M was co-cultured with HPDE6C7 cells for 24 h,which was then treated with CAE and ML-7.The monolayer permeability of HPDE7C7 cells was detected using FITC-Dextran.The mRNA expression of MLCK,ZO-1,Claudin-2,Occludin,Cav1.2 and Cav1.3 was detected by q PCR.The protein expression of MLCK,ZO-1,Claudin-2 and Occludin was detected by Western Blot.The structure of cell microfilaments was observed by direct immunofluorescence assay.Results: 1.Co-cultured with PA,HPDE6C7 cells presented increased permeability,up-regulated MLCK protein and mRNA expression,up-regulated Cav1.2 and Cav1.3 mRNA expression,and down-regulated ZO-1,Claudin-2 and Occludin protein and mRNA expression(P<0.05).The permeability of HPDE6C7 cells increased and local microfilaments were arranged in disorder.2.After stimulation with CAE,the MLCK protein and mRNA expression was further up-regulated,showing statistical significance compared with the PA group(P<0.05).The mRNA expression of ZO-1,Claudin-2 and Occludin was down-regulated,presenting statistical significance compared with the control group(P<0.05).Compared with PA group,the expression of Claudin-2 mRNA decreased,which was statistically s ignificant(P<0.05).The mRNA expression of Cav1.2 and Cav1.3 was up-regulated,without statistical significance compared with the PA group(P>0.05).The protein expression of ZO-1,Claudin-2 and Occludin was down-regulated,with statistical significance compared with the control group(P < 0.05).ZO-1 and Occludin protein expression showed statistical significance compared with the PA group(P<0.05).HPDE6C7 cell permeability increased,normal cell morphology disappeared,and microfilaments depolymerized,and the network structure collapsed.3.After ML-7 intervention,MLCK protein and mRNA expression was down-regulated,with statistical significance compared with the PA + CAE group(P<0.05).The mRNA and protein expression of Cav1.2 and Cav1.3 was pairwisely compared with the PA + CAE group,without statistical significance(P>0.05).The mRNA and protein expression of ZO-1,Claudin-2 and Occludin was up-regulated,and the mRNA and protein expression of ZO-1 and Occludin showed statistical significance compared with the PA + CEA group(P<0.05).The permeability of HPDE6C7 cells decreased,and the arrangement of microfilaments was disordered,and the damage degr ee was less than that of PA+CEA group.Conclusions: 1.CAE up-regulates the protein and mRNA expression of MLCK,and down-regulates the protein and mRNA expression of ZO-1,Claudin-2 and Occludin,and destroy cell microfilament and increases the permeability of HPDE6C7 cells under high fat.2.Inhibiting the expression of MLCK resulted in the decrease of the permeability and the increased expression of ZO-1,Claudin-2,Occludin protein and mRNA of HPDE6C7 cells induced by CAE in the high-lipid culture environment.3.Both PA and PA + CEA can up-regulate the mRNA expression of Cav1.2 and Cav1.3,without statistical significance.ML-7 has no inhibitory effect on the up-regulation.Section III P reliminary study of interfering MLCK gene on the tight junction and permeability of pancreatic ductal epithelial cells in HTGPObjective: Lentivirus-mediated RNA interference was applied for mi RNA interference of the MLCK gene in human pancreatic ductal epithelial cell line HPDE6C7,and a lentiviral vector with EGFP reporter gene was constructed.HPDE6C7 cells were tansfected with the constructed MLCK mi RNA vector to form a stable transfected cell line.The stably transfected cell line was co-cultured with PA and CAE to observe the permeability of single-layer ductal epithelial cells and the expression of TJ-associated protein.Methods: Human MLCK gene was cloned and connected to p Lenti6.3-EGFP vector after enzyme digestion with Asc I and Pme I to construct recombinant plasmid p Lenti6.3-EGFP-MLCK and transfect 293 T cells for package into lentivirus.The lentivirus was used to infect human pancreatic ductal epithelial cell line HPDE6C7.Stable transfected cell lines were screened by blasticidin(BSD),and transfection effect was verified by q PCR.2.The screened MLCK-mir-HPDE6C7 cells,no-load lentivirus negative control group and normal HPDE6C7 cells were co-cultured with PA.After CEA stimulation,the expression of MLCK,ZO-1,Claudin-2 and Occludin protein was detected by Western Blot.The FITC-Dextran method was used to detect the permeability of monolayer HPDE6C7 cells.Results: 1.Sequencing confirmed successful construction of miRNA lentivirus interference vector and recombinant plasmid of human MLCK gene.After HPDE6C7 cells infected by recombinant lentivirus particles produced by 293 T cells were co-transfected,green fluorescent proteins were highly expressed.On the 14 th day after BSD screening,q PCR showed that the mRNA expression of MLCK in the mi RNA lentivirus transfection group decreased significantly than that in the blank control group and the negative control group(P < 0.05).2.The protein expression of MLCK in the L + PA group and the LNC + PA + CEA group was lower than that in other groups(P < 0.05).3.Compared with the control group,MLCK protein expression increased, permeability increased,and Claudin-2,Occludin and ZO-1 protein expression decreased in the LNC + PA group and the LNC + PA + CEA group(P<0.05).4.Compared with the LNC + PA + CEA group,the protein expression of Claudin-2,Occludin and ZO-1 in the L + PA group and(or)the LC + PA + CEA group increased,and cell permeability decreased(P< 0.05).Conclusions: 1.An HPDE6C7 cell line with stable interfering MLCK gene is successfully established.2.After the interference of MLCK gene expression in HPDE6C7 cells,the permeability of pancreatic duct epithelial cell model of HTGP reduces,and the protein expression of TJ-correlated proteins up-regulate. |
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