The Recombinant Adeno-associated Viruses Of The Serotypes 2 Mediated Overexpression Of MnSOD Protects Stria Marginal Cells Of Rat Cochlea Against Oxidation-Induced Apoptosis

PartⅠThe oxidative stress experimental model in hydrogen peroxide induced stria vascularis marginal cells injury in vitroObjective To investigate the oxidative stress experimental model in hydrogen peroxide induced cochlea stria vascularis marginal cells(MCs) injury in vitro. Methods Cultured marginal cells were treated by by 200,300,400,600 and 800umol/L hydrogen peroxide (H2O2) for 0.5,1,2,4,16 and 24h hours. Cell viability was assessed by the CCK-8 assay. The content of the lipid peroxidation production malondialdehyde (MDA )were detected in H2O2 induced marginal cells injury with different concentration H2O2.Apoptosis was assessed by flow cytometry by Propidium iodium staining. The expression of the cleaved Caspase-3 assessed by Western blot.Results Being exposed to H2O2,MCs displayed nuclear pyknosis and margination, cytoplasmic condensation, cell shrinkage and formation of membrane bounded apoptotic bodies.A time-dependent and dose- dependent decrease of cellular viability was deteced with the treatment Of H2O2. Cellular MDA was generated in proportion to the concentration of H2O2 at 2 hours and the number of apoptotic cells increased significantly (P<0.05).Western blot showed the expression of the Cleaved-caspase-3 increased when 200umol/L,300umol/L and 400umol/L H2O2 treated cultured marginal cells. Thereafter the expression of the Cleaved-caspase-3 decreased with 600umol/L H2O2 and with 800umol/L H2O2 the expression of Cleaved-caspase-3 was weak.Conclusion The findings indicated that the experimental model can be established successfully using cultured cells exposed to H2O2 and activation of caspase-3 is associated with Hydrogen peroxide induced the oxidative stress injury of MCs. PartⅡThe recombinant adeno-associated viruses of the serotypes 2 mediated delivery and expression of MnSOD in cultured strial marginal cellsObjectives To obtain transgenic marginal cells(MCs) of the rat cochlea stria vascularis by the recombinant adeno-associated viruses of the serotypes 2 (AAV2) mediated delivery of cDNA of manganese superoxide dismutase (MnSOD).Methods Cultured rat MCs were infected using rAAV2-MnSOD-EGFP at dosage of multiplicity of infection (MOI) 104v.g. /cell and using rAAV2-EGFP as control. The expression of EGFP in MCs was examined using fluorescence microscope every 2 days after transfection.The activity of MnSOD was determinated by colorimetric assays. The expression of MnSOD in MCs was examined using western blot and Laser Scanning Confocal Microscope.Results (1)EGFP expression in MCs could not be detected until 2 days after rAAV2-EGFP transfection and until 4 days after rAAV2-EGFP transfection. The positive EGFP expression in MCs reached fastigium after a week and lasted over a month.(2)According to the results of western blot test and the observation under laser scanning confocal microscope, concentration of MnSOD in the MCs after rAAV2-MnSOD-EGFP transfection was higher than the control cells.Conclusions The rAAV2-MnSOD-EGFP can effectively transfect cultured MCs, and the transgenic cells show a high expression of MnSOD. PartⅢThe Recombinant Adeno-associated Viruses of the Serotypes 2 Mediated Overexpression of MnSOD Protects Stria Marginal Cells of Rat Cochlea against Oxidation-Induced ApoptosisObjective 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) 104v.g. /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 umol/L) for 2 hour and reculturing them in normal medium.After 24h the content of the lipid peroxidation production malondialdehyde(MDA )were detected.Apoptosis was assessed by flow cytometry by Propidium iodium staining. The expression of the cleaved Caspase-3 assessed by Western blot.Results (1)EGFP expression in MCs could not be detected until 4 days after rAAV2- MnSOD-EGFP transfection and reached fastigium after 10days and lasted over a month. The MnSOD level in the rAAV2- MnSOD-EGFP group was higher than the control cells. (2) After Being exposed to H2O2, the content of MDA in rAAV2-MnSOD-EGFP group ,control proup and normal proup were 0.464±0.049,1.103±0.033 and 0.185±0.005 (nmol/mgprot), respectively. the expression of the Cleaved-caspase-3 in rAAV2-MnSOD- EGFP group was lower than control proup and the number of apoptotic cells decreased significantly.Conclusion The results demonstrate the rAAV2-MnSOD-EGFP can effectively transfect cultured MCs, and the transgenic cells show a high expression of MnSOD which can proctect 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 caspas-3.