Authors: Yang Baoxia, Wang Ye, Zhao Xiaowen, Liu Ting, Huang Yusen
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Background
Epithelial-mesenchymal transition (EMT) is one of the pathogenesis mechanisms of posterior capule opcification (PCO). Studying EMT is of important significance for the prevention and treatment of PCO.However, EMT model is lack.
Objective
This study was to establish an in vitro EMT model for the study of human PCO.
Methods
In vitro mimic cataract enucleation was performed on 40 donor eyes, including anterior capsulorhexis, nucleus hydroexpression, and aspiration of lens fibers.The capsular bag of lens was dissected free during the surgery and pinned flat on a plastic culture dish with DMEM/F12 supplemented containing 10% fetal bovine serum for 4 weeks.The proliferation of LECs on the capsular bag was observed by phase-contrast and dark-field microscope in 0, 3, 7, 14 and 28 days after culture.The capsular bag tissues were collected in cultured 0, 3, 7 and 28 days for the preparation of sections and hematoxylin-eosin stain, and the growth and morphology of LECs were examined with optical microscope.The expression and location of α-SMA, E-cadherin and Vimentin were assessed by immunochemistry.The expression levels of α-SMA, E-cadherin and Vimentin mRNA and proteins were detected by using real-time fluorescnce quantitative PCR and Western blot in different time points.
Results
No LECs were seen on the uncultured capsular bag.LECs appeared in cultured day 3 on the periphery capsular bag and grew toward the center and covered the posterior capsule 7 days after cultured, with a cobblestone-like appearance.Wrinkles occurred and extended gradually along with the enhancement of bag tension.Immunochestry showed that the expression intensity of E-cadherin in the capsular bag gradually weakened, and that of α-SMA or Vimentin was gradually enhanced during the culture duration.The relative expression levels of E-cadherhin mRNA at 0, 3, 7, 14 and 28 days after culture were 3.35±0.13, 1.47±0.20, 1.13±0.14, 1.00±0.85 and 0.23±0.03, and relative expression levels of Vimentin mRNA were 1.00±0.73, 1.05±0.14, 2.24±0.43, 2.84±0.34 and 8.57±0.40, and those of α-SMA mRNA were 1.00±0.06, 2.68±0.28, 4.24±0.05, 2.05±0.90 and 15.30±0.19, showing significant differences among different time points (E-cadherhin mRNA: F=23.430, P=0.000; Vimentin mRNA F=8.915, P=0.002; α-SMA mRNA: F=103.500, P=0.000), with the lowest expression levels in the E-cadherhin mRNA and the highest expression levels in the Vimentin mRNA and α-SMA mRNA at 28 days during the culture period (all at P<0.01). The gray values of E-cadherin protein expression were 1.443±0.017, 1.023±0.003 and 0.568±0.018, and those of Vimentin protein were 0.565±0.012, 1.156±0.007 and 1.241±0.009, and those of α-SMA protein were 0.195±0.045, 0.693±0.036 and 1.501±0.005 at 0, 3 and 28 days, with significnant differences among various time points (E-cadherin: F=2 787.000, P=0.000; Vimentin: F=4 488.000, P=0.000; α-SMA: F=1 173.000, P=0.000). The expression levels were significantly declined in E-cadherhin protein and elevated in Vimentin and α-SMA proteins at 3 and 28 days after culture in comparison with before culture.
Conclusions
A novel in vitro EMT model of LECs is established in this study.This model can mimic a natural EMT procedure after extracapsular cataract enucleation and therefore is a useful model for the further research of the mechanism and prevention and treatment of PCO.