| Objective:1.Explore whether ADM induced BXPC-3 epithelial mesenchymal cells into pancreatic cancer ?2.Explore the role of metformin on the transformation of epithelial mesenchymal performed by human pancreatic cancer cells BXPC-3induced by dose of ADM.Method:Adopt the method of ADM in vitro stimulation training BXPC-3cells,EMT model was constructed,and the morphological changes of cells were observed under inverted microscope,Purpose to grouping BXPC-3 cells according to the experiment,can be divided into BXPC-3-CTR group;BXPC-3-ADM group;BXPC-3-ADM + M group;BXPC-3 +M group;Determined by MTT method to detect metformin effects on cell proliferation;Cell scratch repair experiments to detect metformin effects on cell migration;Flow cytometry to detect the influence of metformin on BXPC-3 cell apoptosis;Rt-pcr and Westernblot detection as an important symbol of EMT related molecular p-stat3,Twist,E-cadherin and Vimentin changes at the level of m RNA and protein expression.Results:1.Each experimental cell morphological differences were observed under inverted microscope,ADM BXPC-3 pancreatic cancer cells can be induced by a series of morphological changes.From a typical sample of epithelial cells form to the interstitial cells,cell arrangement is relatively loose and form highly irregular,most cells shows like a fibrous,have the typical morphological changes compare than common BXPC-3 cells.2.Determined by MTT method to detect the change of each cell proliferation,ADM can promote pancreatic cancer Bxpc-3 cells proliferation;Metformin can significantly inhibitpancreatic cancer Bxpc-3 cells proliferation;and metformin inhibits ADM for cell proliferation promoting effect(P < 0.01).3.Comparing the experimental cell migration ability by scratch repair experiment,BXPC-3-ADM groups compared with BXPC-3-CTR groups,24 hours after the scratch,BXPC-3-ADM groups has a small gap than BXPC-3-CTR cell,48 hours after the scratch,BXPC-3-ADM groups the gap of scratchs has narrowed and gradually close to state of healing,while control grop scratchs gap is still relatively wide.No matter 24 hours or 48 hours after the scratch,BXPC-3-ADM groups has a small gap than BXPC-3-ADM+ M cell and BXPC-3-CTR groups has a small gap than BXPC-3+M cell.4.Flow cytometry to detect the changes of cell apoptosis,ADM can inhibit apoptosis;Metformin can promote pancreatic cancer Bxpc-3cells apoptosis,and metformin inhibits ADM inhibition of apoptosis(P <0.01).5.PCR to detecting experimental cell EMT related molecular mRNA expression level,the PCR results showed that compared with the control group,BXPC-3 cells treated with ADM E-cadherin m RNA expression decreased obviously,and Twist,Vimentin m RNA expression significantly higher(p < 0.01).Compared with the BXPC-3-ADM group,BXPC-3-ADM+M group and BXPC-3+M group E-cadherin m RNA expression increased,while Twist and Vimentin m RNA expression reduced(p < 0.05).6.Western blot is used to detect the expression level of each experimental cells related with EMT sign molecules protein,the results showed that compared with the control group,BXPC-3 cells treated with ADM E-cadherin protein expression quantity decreased,and P-stat3,Twist and Vimentin protein expression is significantly higher(p <0.05).Compared with the BXPC-3-ADM group,BXPC-3-ADM+M group and BXPC-3+M group E-cadherin protein expression quantity increases,while P-stat3,Twist and Vimentin protein expression were lower(p < 0.01).Conclusion:1.ADM can induce pancreatic cancer BXPC-3 cells produce EMT,also improve the cell proliferation and migration ability,well as enhance the expression level of EMT related marks molecules produces.2.Metformin can significantly inhibit pancreatic cancer BXPC-3 cells proliferation and invasion ability,inhibit tumor cells EMT process at the same time. |
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