Objective To observe the effect of epithelial-mesenchymal transdifferentiation (EMT) of human retinal pigment epithelial (RPE) cells induced by vitreous humor in vitro. Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10% serum containing Dulbecco minimum essential medium (DMEM)/F12 medium (group A), or the same medium supplemented with 25% human vitreous (group B). The morphological changes were observed with a phase contrast microscrope. Cell migration, invasion and contractility were tested using a scratch wound assay, Transwell invasion assay and collagen gel contraction analysis. The expression levels of alpha;-smooth muscle actin (SMA) and Snail1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The RPE cells in group A were flat and gathered together. The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge, or spindle-shaped, and appeared to separate. In group A, filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape. In group B, filamentous actin reorganized and formed fan-like flat pseudopodia at one edge of the cells. Compared to group A, the migration and invasion of the cells increased significantly (t=14.190, 22.630; P＜0.05), but contractility decreased remarkably (t=6.221, P＜0.05) in group B. Compared to group A, the expression level of Snail1 mRNA increased significantly (t=3.218, P=0.032), but the expression level of alpha;-SMA mRNA decreased (t=3.990, P=0.016). Conclusions Vitreous humor can induce the EMT of RPE cells. Increasing cell migration, cell invasion, and expression of Snail1 mRNA as well as up-regulated cellsprime; contractility and expression of alpha;-SMA mRNA may be the mechanism.
ObjectiveTo study the role of Rac1 in the epithelial-mesenchymal transition (EMT) process of retinal pigment epithelial cells (RPE) induced by transforming growth factorβ(TGF-β). MethodsHuman ARPE-19 cells were divided into 4 groups including control group, TGF-βgroup, TGF-β+NSC23766 group, NSC23766 group. NSC23766 was added to medium 2 hours before TGF-βtreatment to block the Rac1 receptors.α-smooth muscle actin (α-SMA) expression was measured by immunofluorescence and Western blot. Cell scratch assay, invasion assay and gel contraction experiments were used to measure cell migration, invasion, cell contraction. ResultsThe expression ofα-SM A was higher in TGF-βgroup, compared with the control group, TGF-β+NSC23766 group (F=825.314, P < 0.05). Cell scratch assay showed that the cellular gap was less in GF-βgroup, compared with the control group, TGF-β+NSC23766 group, NSC23766 group (F=177.351, P < 0.05). Cell invasion assay showed that, the number of cells pass through the fiber membrane was the same in TGF-βgroup and other 3 groups (F=0.371, P=0.055). Gel contraction assay showed that TGF-βcan promote the cellular contraction, compare to the control group, TGF-β+NSC23766 group, NSC23766 group, the difference was statistically significant (F=40.473, P < 0.05). ConclusionRac1 play a role in TGF-β-induced behavioral changes of RPE cells; NSC23766 inhibit RPE cellular behavior change by regulating Rac1 activation.