Molecular Characteristics of Primary Pediatric Lens Epithelium Cells
Daniel J. Salchow, MD; Laura Wernecke; Eckart Bertelmann, MD; Nadine Reichhart, PhD; Olaf Strauss, PhD
Charite – University Medicine Berlin
Introduction: After-cataract is a common complication of cataract surgery in children. Epithelial-to-mesenchymal transformation (EMT) of lens epithelium cells is thought to be involved in after-cataract formation. To better understand the molecular basis of these processes, we studied the molecular characteristics of PLEC and of a lens cell line (HLE-B3) before and after stimulation with transforming growth factor beta2 (TGFbeta2), which has been implicated in EMT and after-cataract formation.
Methods: PLEC were obtained during pediatric cataract surgery by harvesting anterior capsules, and were cultivated in DMEM/F-12. HLE-B3 cells were cultivated in EMEM medium supplemented with 20% fetal calf serum and 1% penicillin-streptomycin. We studied the expression of the genes for crystalline alpha (CRYAB) and connexin 43 (Cx43) as epithelial markers, and for fibronectin (FN1), integrin alpha V (ITGAV), alpha smooth-muscle actin (aSMA) and collagen 1 alpha 2 (COL1A2) as mesenchymal markers. Expression profiles before and after stimulation with TGFbeta2 were examined using reverse transcription (RT), quantitative real-time (qRT) PCR and gel electrophoresis.
Results: The protocol for cultivating PLEC was established. Expression of CRYAB (in HLE-B3, p= 0.0003), ITGAV (in HLE-B3, p= 0.01), COL1A2 and FN1 (in pLEC, p= 0.003 and HLE-B3, p= 0.0002) were significantly upregulated after stimulation with TGFÃŸ-2. Total gene expression levels were significantly higher in PLEC compared to HLE-B3. Patient age at surgery and cataract morphology affected growth of PLEC in culture.
Discussion: Compared with HLE-B3, PLEC showed higher overall gene expression levels. Stimulation of PLEC and HLE-B3 cells with TGFbeta2 led to a shift in the expression profile, compatible with EMT. EMT is involved in the development of after-cataracts.
Conclusion: Using cell culture, gene expression profiles of PLEC can be studied. This model may be useful to study mechanisms of after-cataract development, its prophylaxis and treatment.
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