ZO-1,E-cadherin And Connexin 43 In Retinal Pigment Epithelial Cells In The Early Stage Of Proliferative Vitreoretinopathy And Their Internal Linkage And Mechanism

Purpose:Mature retinal pigment epithelial(RPE)cells are at rest under physiological conditions.The cells are regularly arranged between cells to form a single layer of epithelial tissue sandwiched between the neural retina and the choroid to maintain neural retina function.The integrity of its epithelium is maintained by the interaction of three types of cell connections(tight,anchored,and gap junctions)between adjacent cells and between cells and the basement membrane.The three signature connexins are zona occludens 1(ZO-1),E-cadherin(E-Ca)and connexin 43(CX 43).Proliferative vitreoretinopathy(PVR)is the main reason for the failure of retinal detachment surgery,and the failure to find a definite clinical treatment has become a problem in the field of ophthalmology.An important mechanism in the pathogenesis of PVR is that free retinal pigment epithelial RPE cells have undergone an epithelial-mesenchymal transition(EMT).However,before EMT occurs in RPE cells,the cells first lose contact,that is,the intercellular connexins are damaged or abnormally expressed.However,in the PVR microenvironment,the early changes of RPE intercellular connexins and their inherent correlations and mechanisms have not been thoroughly studied.This study will investigate the linkage changes of three connexins E-Ca,CX 43 and ZO-1 in RPE cells before EMT occurs in RVR cells;clarify the mechanism of RPE cells losing normal contact before EMT.Method:(1)Take the donated eyeballs for corneal transplantation,extract and culture human primary RPE cells(phRPE),and treat phRPE cells with 10 ng / ml TGF-?1 at different times of 0h,1.5h,3h,6h,12 h,etc.After the gradient,the expression of E-Ca,CX 43 and ZO-1 in cells in the early stage of TGF-?1 action was detected by Western blotting.(2)Design and synthesize specific siRNA for E-Ca,CX 43 and ZO-1 proteins respectively.Transfect siRNA into phRPE cells to knock down the expression of E-Ca,CX 43 and ZO-1.Detect the knockdown efficiency by Western blotting and real-time quantitative PCR and observe the expression changes of the other two proteins to describe these three Regulatory network between proteins.(3)Take the retinas of C57 BL / 6 mice and observe the ultrastructure of the intercellular connection in RPE of mice with transmission electron microscope(TEM).(4)Isolate the mouse retinal pigment epithelium-Bruch 's membrane-choroid complex(RBCC),and after depigmentation,mount the plates for immunofluo rescence staining to observe the expression and localization of the three intercellular connexins.(5)Construction of E-Ca and Cx 43 overexpression plasmids,overexpression of E-Ca and CX 43 in ARPE-19 cells,followed by co-immunoprecipitation with antibodies to E-Ca and Cx 43,respectively,using WB The expression of the other two proteins in the co-precipitate was examined to determine whether they had a direct interaction.(6)Use siRNA to knock down E-Ca,CX 43 and ZO-1 in ARPE-19 cells,and then carry out fluorescent yellow dye transfer experiment to detect the change of ARPE-19 cell gap junction function.In the same way,knock down the three proteins of phRPE cells and use transwell to conduct FITC-dextran cell permeability test to observe the changes of epithelial barrier function.Result:(1)The expression levels of E-Ca,CX 43 and ZO-1 protein of phRPE cells after TGF-?1 treatment gradually decreased within 12 h,and among these three proteins,E-Ca was the first protein to be down-regulated.(2)The knockdown of any of the three proteins E-Ca,CX 43 and ZO-1 will cause the down-regulation of the other two proteins.(3)Transmission electron microscopy found that there are three kinds of connection structures between mouse RPE cells: adhesive connection(adhesive tape,desmosome),gap connection and tight connection close to each other.(4)Immunofluorescence detection of RBCC in mice showed that E-Ca,CX 43 and ZO-1 co-localized in RPE cells.(5)Co-immunoprecipitation found that the three connexins have a direct interaction binding relationship with each other.(6)Knocking down any of E-Ca,CX 43 and ZO-1 protein will damage the gap junction function and epithelial barrier function of RPE cells.Conclusion:Studies have shown that the expression of three connexins E-Ca,CX 43 and ZO-1 between RPE cells under PVR microenvironment(TGF-?1)before EMT is down-regulated,and E-Ca is the earliest down-regulated protein.E-Ca,CX 43 and ZO-1 regulate and combine with each other to form an intercellular junction complex and maintain the structure of intercellular junctions.