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blanca elizabeth martinez baez, Rosario Gulías-Cañizo, Valeria Sanchez-Huerta, Federico Castro Muñoz-Ledo, Lorena Zendejas Reyes; Evaluation of the effects of RB-35 on the viability and growth of a corneal epithelium cell line. Invest. Ophthalmol. Vis. Sci. 2017;58(8):967.
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RB-35 has shown to possess antioxidant properties due to its free radical scavenging activities and its protective effects against lipid peroxidation. Since oxidative damage plays a major role in many ocular surface diseases, we aimed to evaluate the effects of RB-35 in a corneal epithelium cell line as preliminary evidence of its therapeutic potential.
A rabbit corneal epithelium cell line was used, RCE-1(5T5), which reproduces in vitro the differentiation and proliferation processes normally present in primary cultures of corneal epithelium.The experimental strategy included: Colony forming assay to evaluate cell growth, flow cytometry to evaluate cell viability and immunofluorescence to evaluate cell phenotype. All experiments were performed in triplicate.In the colony forming assay, the cells are seeded with the standard density for a 35 mm plate (3 x 104) on day 0; on day 1 control cells receive corneal medium + EGF and treated cells receive corneal medium + EGF + RB-35 at different concentrations (10 µM, 20 µM, 30 µM, 50 µM, 100 µM). On day 3, cells are harvested and reseeded with a seeding density of 500 cells per 60 mm plate, and the growth is evaluated on a daily basis until the culture forms colonies. At this point, colonies are stained with rhodamine.To evaluate cell viability,we used 7-aminoactinomycin D, a fluorescent intercalator that undergoes a spectral shift upon association with DNA, which stains non-viable cells. These experiments were analyzed by flowcytometry.We also performed the immunofluorescence staining protocol for keratin 3, a marker of terminal differentiation specific of corneal epithelial cells, to evaluate cell characteristics.
We demonstrate a colony forming efficiency similar to the control, which rules out an anti-proliferative effect on cell growth. The percentages of colony forming efficiency between control cells and treated cells did not show statistical significance. Moreover, in the viability assay, we observed a statistical significant difference favoring RB-35.
RB-35 does not induce cytotoxic effects in a concentration range of 10 to 100 µM, and it demonstrates a protective effect evidenced by reduction of apoptosis without inducing cell phenotypic changes. These results give the first positive evidence to continue evaluating RB-35 as a potential treatment for ocular conditions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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