The recombinant adeno-associated viruses of the serotypes 2 mediated overexpression of MnSOD protects against oxidation-induced apoptosis in stria marginal cells of rat cochlea.
- Author:
Jun LI
1
;
Weijia KONG
;
Xueyan ZHAO
;
Yang YANG
;
Yujuan HU
Author Information
1. Department of Otorhinolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 400022, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Apoptosis;
Caspase 3;
metabolism;
Cells, Cultured;
Cochlea;
metabolism;
Dependovirus;
genetics;
Oxidative Stress;
Rats;
Rats, Wistar;
Stria Vascularis;
cytology;
Superoxide Dismutase;
biosynthesis;
Transfection
- From:
Journal of Clinical Otorhinolaryngology Head and Neck Surgery
2011;25(10):463-468
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To investigate the influence of overexpression of manganese superoxide dismutase (MnSOD) of stria marginal cells (MCs) of the rat cochlea by the recombinant adeno-associated viruses of the serotypes 2 (AAV2) mediated gene-delivery for hydrogen peroxide-induced oxidative stress in vitro.
METHOD:Primary cultures of MCs were infected using rAAV2-MnSOD-EGFP at dosage of multiplicity of infection (MOI) 10(1)v x /cell and using rAAV2-EGFP as control. The expression of MnSOD in MCs was examined using western blot and the activity of MnSOD was determinated by colorimetric assays. Oxidative stress was induced in MCs by exposing them to H2O2 (400 micromol/L) for 2 hour and preculturing them in normal medium. After 24 h the amount of the lipid peroxidation production malondialdehyde (MDA) was detected. Apoptosis was assessed by flow cytometry by Propidium oidium staining. The expression of the cleaved Caspase-3 was assessed by Western blot.
RESULT:(1) EGFP expression in MCs could not be detected until 4 days after rAAV2- MnSOD-EGFP infection and reached fastigium after 10 days and lasted over a month. The MnSOD level in the rAAV2- MnSOD-EGFP group was higher than that in the control group. (2) After being exposed to H2O2, the amounts of MDA in rAAV2-MnSOD-EGFP group, control group and normal group were 0.464 +/- 0.049, 1.103 +/- 0.033 and 0.185 +/- 0.005 (nmol/mg prot), respectively. The expression of the cleaved-caspase-3 in rAAV2-MnSOD-EGFP group was lower than that in control group and the number of apoptotic cells decreased significantly.
CONCLUSION:The results demonstrate that the rAAV2-MnSOD-EGFP can effectively transfect cultured MCs, and the transgenic cells show a high expression of MnSOD which can protect the MCs against oxidative challenge. The role of overexpression MnSOD in MCs apoptosis induced by oxidative injury may be associated with suppressing the activation of caspase-3.
