Graduation Year

2021

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Graduate School

Major Professor

Manas R. Biswal, M.F.Sc., Ph.D.

Committee Member

Vijaykumar Sutariya, M.Pharm., Ph.D., R.Ph.

Committee Member

Radouil Tzekov, , MD., Ph.D.

Committee Member

Kevin Nash, Ph.D.

Keywords

antioxidants, glutathione, reactive oxygen species

Abstract

Purpose: Loss of Retinal Pigment Epithelial (RPE) cells and photoreceptors leads to Age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly worldwide. Oxidative stress-mediated damage is one of the factors that impair the survival of RPE and photoreceptors in dry-AMD. We sought to determine the role of human Glutathione S-Transferase Mu-1 (GSTM1) in protecting RPE cells from oxidative stress-induced changes.

Method: We cloned human GSTM1 cDNA with P2A tag into a lenti-plasmid vector and later made a lentivector to express GSTM1. After that, we infected human retinal pigment epithelial cells (ARPE-19), screened stable ARPE-19 cells expressing GSTM1 with supplementation of Puromycin antibiotic in the media, and performed western blotting to check GSTM1 expression in stable cells. The oxidative stress was induced by H2O2 on stable and control ARPE-19 cells and MTT assay and flow cytometry was employed to study cell viability. Real-time PCR (RT-PCR) was used to measure changes in the expression of other antioxidant genes in response to GSTM1 gene augmentation. For immunocytochemical analysis, we stained differentiated RPE cells with ZO1 junctional protein and fluorescence images were analyzed to evaluate the cellular integrity.

Results: The western blotting with GSTM1 antibody confirmed the expression of exogenous GSTM-1 protein and further affirmed by P2A antibody. The MTT (n=6) and flow cytometry assay (n=3) demonstrated significant cell viability compared to the control cells (p< 0.05) in response to H2O2 (200μM and 500μM) induced oxidative stress. The RPE junctional integrity was maintained in the GSTM1 expressing differentiated cells as compared to the controls. RT-PCR analysis demonstrated moderate induction in the expression of cytoprotective genes.

Conclusion: GSTM1 gene augmentation in RPE cells can protect against oxidative stress-mediated changes. Our research opens an avenue for a potential gene therapy strategy to reverse oxidative stress-mediated changes, thus preventing RPE cell death associated with progressive vision loss in Dry-AMD.

Download

Share

COinS