Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Autor: Bascuas Castillo, Thaïs, Kropp, Martina, Harmening, Nina, Asrih, Mohamed, Izsvák, Zsuzsanna, Thumann, Gabriele
Rok vydání: 2020
Předmět:
pigment epithelium-derived factor
Cell Survival
retinal pigment epithelium
Cell Count
Retinal Pigment Epithelium
Transfection
Antioxidants
Cell Line
Sleeping Beauty transposon system
Humans
Uncoupling Protein 2
Nerve Growth Factors
immunofluorescence
glutathione
antioxidant cell protection
Phosphorylation
Eye Proteins
age-related macular degeneration
in vitro assay
cell viability
Cells
Cultured
Serpins
western blot
Cell Death
Granulocyte-Macrophage Colony-Stimulating Factor
Epithelial Cells
Hydrogen Peroxide
Glutathione
eye diseases
Neuroprotection
Tissue Donors
ddc:616.8
Oxidative Stress
transfection
Gene Expression Regulation
Oxidative stress
granulocyte-macrophage colony-stimulating factor
Culture Media
Conditioned
gene expression
DNA Transposable Elements
ELISA
sense organs
Proto-Oncogene Proteins c-akt
Biomarkers
Zdroj: Journal of Visualized Experiments, No 166 (2020) P. e61957
ISSN: 1940-087X
Popis: Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.
Databáze: OpenAIRE
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