Preliminary Study On The Effects Of Triglyceride On Proliferation,Apoptosis,MLCK And Cell Adhesion Molecule Expression Of Pancreatic Duct Epithelial Ceels

Objective To investigate the effects of different concentrations of triglyceride on proliferation and apoptosis of pancreatic duct epithelial cells.Whether triglyceride levels affect the expression of genes and proteins of MLCK,affect the gene expression of ICAM-1,desmocollin-3,?-catenin and E Cadherin;Whether the knockout of MLCK gene affects the gene expression of cell adhesion molecule ICAM-1,desmocollin-3,?-catenin and E Cadherin,and whether MLCK mediates the expression of pancreatic duct mucosal adhesion molecule.Methods Human pancreatic duct epithelial cells were used as the model and cultured at different levels of triglycerides.Triglycerides with different concentration gradients were divided into 6 groups(0.5mM,1.0 mM,2.5 mM,5.0 mM,10.0 mM).After culture for 48 hours,the changes of cell morphology and intercellular contact distance were observed by inverted microscopy.Cck-8 cell flow assay was used to detect the effects of different triglyceride levels on proliferation and apoptosis of human pancreatic duct epithelial cells.The mRNA expression of MLCK,ICAM-1,desmocollin-3,?-catenin and E Cadherin in cells cultured with different concentrations of triglycerides was determined by real-time fluorescence quantitative PCR.Western blot was used to detect the protein expression of MLCK in cells cultured with different concentrations of triglycerides.Construction of stable transfected cell lines(MLCK-mirRNA)by silencing MLCK gene with lentivirus mediated miRNA interference with hpde6-c7 cell line,Construction of stable transfected hpde6-c7 cell lines by negative virus transfection(NC-mirRNA),Groups of HPDE6-c7,NC-mirRNA and MLCK-mirRNA cells were treated with triglyceride at different concentrations.HPDE6-c7(0?1.0 mM?2.5 mM),NC-mirRNA(0?1.0 mM?2.5 mM),MLCK-mirRNA(0?1.0 mM?2.5 mM),The mucosal barrier related protein gene expression of MLCK icam-1 Desmocollin3 beta-catenin E Cadherin was determined by real-time fluorescence quantitative PCR.ResultsThere was no significant change in cell morphology when TG concentration was 0.5mM,with the increase of TG concentration,the number of apoptosis of HPDE6-c7 cells increased under the microscope,and the cells became thinner and longer,showing changes of cell fibrosis damage and increasing cell spacing.The effect of triglyceride level on the activity of HPDE6-c7 normal cells was measured by cck-8 method.The results showed that: low triglyceride level did not significantly inhibit the proliferation activity of HPDE6-c7 cells,while high triglyceride level(2.5,5.0,10mM)significantly inhibited the activity of cells,which was statistically different from the normal control group(P <0.05),And a dose-dependent performance.The effect of triglyceride level on the apoptosis of HPDE6-c7 normal cells was measured by cell flow technique,and it was found that the addition of low concentration(0.5mm 1.0mm)triglyceride did not cause the apoptosis of HPDE6-c7 cells compared with the normal group,High concentration(2.5mm 5.0mm 10mm)of triglyceride promoted apoptosis,and there were statistically significant differences between the two groups compared with the control group(P <0.01).With the increase of triglyceride concentration,the expression of MLCK gene and protein in HPDE6-c7 cells could be up-regulated in a dose-dependent manner,and the pairwise comparison between the two groups showed statistical differences(P <0.05).Triglyceride can up-regulate the gene expression of ICAM-1,?-catenin and E Cadherin and down-regulate the expression of desmocollin-3.MLCK gene was silenced by lentivirus mediated miRNA interference with HPDE6-c7 cell line,The results showed that HPDE6-c7 and NC-mirRNA cell lines significantly up-regulated the gene expression of MLCK with the increase of triglyceride concentration,After silencing MLCK gene expression,there was no significant change in MLCK expression with the increase of triglyceride concentration.The expression of ICAM-1 was up-regulated with the increase of triglyceride concentration of HPDE6-c7?NC-mirRNA ? MLCK-mirRNA,After silencing the expression of MLCK,ICAM-1significantly increased.HPDE6-c7 and NC-mirRNA cell lines significantly up-regulated the expression of ?-catenin and E Cadherin with the increase of triglyceride concentration,and there were statistically significant differences between different concentrations(P <0.05).After silencing the expression of MLCK gene,there was no significant change in the expression of-catenin E Cadherin with the increase of triglyceride concentration(P >0.05).All the three cells down-regulated the expression of desmocollin-3 with the increase of triglyceride concentration,Compared with normal hpde6-c7 cells,the expression of DSC-3 in,NC-mirRNA and MLCK-mirRNA cells were more significantly down-regulated,and the pair-to-paircomparison between the two groups showed statistical differences(P<0.05).Conclusions1.High concentration of TG has cytotoxic effects on hpde6-c7 cells,which can cause fibrosis and affect the balance of cell proliferation and apoptosis.2.TG up-regulates the gene expression of MLCK,ICAM-1,?-catenin and E-Cadherin,down-regulates the gene expression of DSC-3,which synergistically leads to the impairment of PDMB barrier function.