The Effect Of PI3K/AKT Pathway On EMT Of Human RPE Cells

Retinal detachment(RD)is a kind of serious disease that can cause blindness.Its pathologic manifestation involves separation of retinal neuroepithelial layer and pig ment epithelium layer.RD is induced by the whole layer of retinal hole,vitreous tracti on and retinal exudates.In recent years,with the application of vitrectomy and other sur gical methods,the cure rate of retinal detachment surgery had been improved.At pres ent,the majority of retinal surgery achieves the anatomical reduction but the poor visu al acuity remains recrudescence after surgery.How to improve the postoperative visual acuity and reduce the recurrence rate of retinal detachment are urgent problems to be solved.Proliferative vitreo retinopathy(PVR)is closely related to the incidence of retinal detachment.Studies have indicated that one of the important cause of PVR is epithel ial mesenchymal transition(EMT)of retinal pigment Epithelial(RPE)cells.Epithelial mesenchymal transition is the cellular basis of PVR,which is related to various cytoki nes and cell pathways.The phosphatidylinositol 3-kinase/serine threonine protein kinas e(PI3K/Akt)signaling pathway is an important pathway mediated RPE cells to the E MT.Transforming growth factor-beta2(TGF-?2)is found in RD,which is duced fro m RPE cell to EMT transformation,and it is the cytological basis of PVR.PI3 K /Akt p athway is one of the most important pathways.More and more studies suggest that mechanical stress(MS)plays an important rol e in cell proliferation,differentiation,apoptosis,gene expression and organization grow th,physiological process of integration,and some physiological and pathological proces ses.These processes are similar to the PVR processes which are caused by RPE cells abnormal proliferation.In this process,RPE cells are pulled by mechanical stress.Ho wever,the mechanism of the activation of retinal cells induced by excessive mechanic al stress has not been clarified.Flexcell tissue mechanical culture system can not only provide tensile stress,compressive stress and shear stress loading,but also provide te nsile stress and shear stress hybrid force and the load of a variety of 2D and 3D tiss ue.Flexcell tissue mechanical culture system can lay not only mechanical stress stimul ation on cells and tissues,but also be used in the three-dimensional culture,artificial biological organization construction and dynamic simulation.Flexcell tissue mechanical culture system can not only single axial to pull the extension,but also can biaxial to stretch.Flexcell tissue mechanical culture system can not only adjust the magnitude of the force ccording to the need,but also simulate different stress time.Therefore,Fl excell Device of Mechanical Stress excessive stress in vitro stimulaes RPE cells to in duce RPE cells to EMT more accurate and produce persistent mechanical stretch from fibrous proliferative membrane,and to produce the pathophysiological process of PV R.In this paper,making use of mechanical traction and TGF-?2 induced RPE cell to EMT,the expression changes of PI3 K,PPI3K,AKT and PAKT were observed in t he PI3K/Akt pathway,as well as the EMT related protein(vimentin,alpha-SMA)relate d changes,to explore their relationship.Part1 : Effects of Mechanical Stress on the PI3K/AKTPathway of Human RPE Cells in the first partObjective:The mechanism of retinal detachment is not clear,this paper aims to study volume changes of supernatant IL-8 and the changes of of PI3 K / Akt pathway in human RPE cells which were stretched by the Device of Mechanical Stress.Methods:Human RPE cells were stretched by the Device of Mechanical Stress f or 0 h,1 h,3 h,6 h,9 h.Labeled as the control group,1 h group,3 h group,6 h grou p,9 h group.The changes of IL-8 secreted by human RPE cells in different experim ental groups were detected by ELISA.Expression levels of PPI3 K,T-AKT,PI3 K PAKT were observed by the immunofluorescence and Western blot method.Results:With the extension of stretch time,the volume of supernatant IL-8 in d ifferent groups increased gradually [924.79±5.92 pg/m L?947.73±5.34 pg/mL?974.53±5.74 pg/m L?979.57±1.12 pg/mL?1019.22±4.25pg/mL].The results reviewed that the dif ferences between different groups were significant(F=166,p<0.01).The differences betwe en stretch groups and control group were significant(P < 0.05).The protein expressio n of PAKT increased [0.61±0.02?0.97±0.05?0.99±0.04?1.21±0.11?1.20±0.07].The d ifferences between different groups were significant(F=41.24,P < 0.01).The differenc es between stretch groups and control group were significant(P < 0.05).Conclusion:With the extension of stretch time,the volumes of supernatant IL-8 in different groups increased gradually and the PI3 K / Akt pathway was activated in human RPE cells which was stretched by the Device of Mechanical Stress.The result s offer a method to explore the reasons of EMT and provide a basis for the preventio n and treatment of PVR.Part2:Effects of LY294002 on EMT Induced by TGF-?2 in RPE CellsObjective: To study the changes of EMT in RPE cells induced by TGF-?2 aft er inhibiting the pathway of PI3K/AKT specific inhibitors.Methods: 10 ng / ml TGF-?2 were supplement in RPE cells.30 umol / L LY294002 was added to observe RPE cells morphological changes in different time(0d,1d,2d,3d).Expression levels of P-AKT ?vimentin and-SMA were observed by the imm unofluorescence and Western blot method.Results: The experimental group with 30umol/L LY294002 showed a significant decrease of the protein PAKT expression at 3 hours and 6 hours,and the inhibition ef fect was not obvious after 24 hours.P-AKT in the control group,the experimental gr oup of 3 hours group,6 hours group than the control group,the difference was statist ically significant(p<0.05),the 24 hours group compared with the control group,the d ifference was not statistically significant(P>0.05).With the induction time of TGF-be ta 2 prolonged,the cell structure in the marginal RPE cells was disordered,and the tr ansition to EMT was obvious.The degree of EMT increased with time.The experime ntal groups compared with the control groups were less transition to EMT.The results of immunofluorescence staining showed that vimentin and-SMA decreased significant ly in the experimental group.Conclusion: TGF-?2 can induce the change of RPE cells to EMT,which is corr elated with the induction time,and LY294002 can decrease the RPE cell to EMT tran sformation.