Research On Transdifferentiation Of Human Retinal Pigment Epithelium Subcultured In Vitro

Objective: To investigate the effect of serial subcultivation on the retinal pigment epithelium in vitro.Methods: The cellular morphology of human retinal pigment epithelium was observed by invert microscope and the their expressions of keratin andα-smooth muscle actin were detected by immunocytochemistry stain. The capability of different passages of human retinal pigment epithelium inducing lymphocytes apoptotic and their expression of Fas was observed by flow cytometry.The function of different passages of human retinal pigment epithelial cells phagocytizing pigment granules was observed by Multikcan Ascen Labsystems.Results: During routine culture on tissue culture plastic, human retinal pigment epithelium lose epithelial characteristics and acquire a mesenchymal cell-like phenotype. The cells transdifferentiated when continued passage in vitro. This correlated with the loss of the epithelial morphology, decreased expression of the epithelial marker cytokeratin 18, redistribution of the actin cytoskeleton and de novo expression ofα-smooth muscle actin. The expression of cytokeratin 18 was negative correlation with continued passage (r = -0.831,P < 0.001) and that ofα-smooth muscle actin was positive correlation with continued passage(r =0.456, P =0.002). When co-cultured with lymphocytes,the expression of Fas was tapering and the duration of culture in vitro were significantly correlated with the expression of Fas(The rate of positive cell: r = -0.859,P <0.001, The mean fluorescence intensity: r = -0.880,P <0.001).The rate of apoptosis of lymphocytes was taper going down to posterity,and the of culture in vitro were significantly correlated with the rate of apoptosis of lymphocytes(r =-0.838 ,P < 0.001).The expression of Fas and the rate of apoptosis of lymphocytes quickly descend going down to posterity.There was no difference between third passage and fourth passage(P>0.05).The capability of swallowing pigment were tapering,and it quickly descend from the third passage.The absorbance of human retinal pigment epithelium were significantly correlated with the duration of culture in vitro(r = -0.892,P < 0.001),and the third passage was different from the fourth(P < 0.05).Conclusions: The more passage numbers,the biger cytometaplasia.The small quantity of the origin and the first passages of human retinal pigment epithelialium restrict the development of HRPE transplantation.The second and the third passages of human retinal pigment epithelialium are almost the same with the origin cells on the morphous and the function,and the quantity of the cells is enough for the experiment of HRPE transplantation.So the second and the third passages of human retinal pigment epithelialium are suitable for the HRPE transplantation.