The Effect Of Overexpression Of PGC-1α On The MtDNC4834Common Deletion In A Rat Cochlear Marginal Cell Senescence Model

Objective: Primary culture of cochlear marginal cells (MCs) in neonatal SD rats andestablished the cochlear marginal cell senescence model with mtDNA4834commondeletion (CD).Methods: The bilateral temporal bones of neonatal (3days old) SD rats were removed andthe strias vascularis from the apical turn to basal turn were dissected. After digesting by theenzyme, the MCs were resuspended and plated in cell culture plates with Epithelial CellMedium-animal (EpiCM-animal), which were placed in an incubator at37oC in5%CO2and95%air. Observe the morphology of MCs under a microscope and identify the MCs byimmunohistochemistry. Treated the MCs with D-galactose for48hours with differentconcentration levels and select the optimal concentration level by CCK-8. Under thisconcentration, treated the MCs with different time duration, detected their mtDNA4834common deletion (CD) rates by real-time polymerase chain reaction (RT-PCR) and selectedproper time duration. Using β-galactosidase (SA-β-gal) staining kit, detected the aging degree of MCs being treated under the optimal concentration and time duration.Results: MCs will grow stick to the wall after plated onto the culture plates for24hours.MCs are closely connected and may grow into a monolayer with a “cobblestone-like”appearance under a microscope. The secreting bubbles can be seen on the top of the cell,and the “dome” can be also observed. The fibroblasts can be removed by low serumconcentration culture medium, difference to stick wall and selective digestion. Furthermore,a high CK18(a biomarker of the epithelium) fluorescent signal could be detected in thecultured MCs. Detecting by CCK-8, MCs activities were suppressed significantly whentreated with D-galactose at14mg/ml(p <0.05) and16mg/ml (p <0.01). So we chose the12mg/ml, the previous concentration level, to treated the MCs. Detecting the CD ratesunder this concentration, we found the CD rates at120h（r0.01<p <0.05），168h（rp <0.01）and216hr（p <0.01）had significantly increase compared with their control groupsrespectively. The MCs treated with12mg/ml D-galactose for120hr showed positiveSA-β-gal staining compared with the control group.Conclusion: Primary culture of strias vascularis marginal cells can be successfully doneand the purified marginal cell line was established. Meanwhile treated with12mg/mlD-galactose for120hours, an MC senescence model harboring CD was successfullyestablished. Objective: The expression and the distribution of PGC-1α, NRF-1, Tfam, NF-κB, FOXO-1and SIRT-1in stria vascularis marginal cells. Research the conditions for successfullytransfecting the PGC-1a-GFP-Ad into MCs.Methods: Using an immunofluorescence method and a laser-scanning confocal microscope(LSCM), we detected the distribution of PGC-1α, NRF-1, Tfam, NF-κB, FOXO-1andSIRT-1in the MCs. Screen the proper titre for tranfecting MCs with recombinantadenovirus by virus titres review experiments and the MCs infection pretests.Results: In MCs the PGC-1α and NRF-1were mainly expressed in the nucleus. Tfam wasexpressed in both the cytoplasm and nucleus while NF-κB was also expressed in both thenucleus and cytoplasm, but to a lesser extent in the cytoplasm. FOXO-1was expressed inboth the nucleus and cytoplasm, and SIRT-1was mainly expressed in nucleus and alsoexpressed in cytoplasm. The optimal adenovirus titre for tranfecting MCs with recombinantadenovirus is4×107PFU/ml which is being selected by virus titres review experiments andthe MCs infection pretests. And the MCs will present green fluorescence for labeling theGFP.Conclusion: The PGC-1α, NRF-1, Tfam, NF-κB, FOXO-1and SIRT-1expressed in MCswith different distribution. Achieve the optimal adenovirus titre for tranfecting MCs andsuccessfully transfected recombinant adenovirus into MCs. Objective: To investigate the protective roles and regulation mechanisms of PGC-1α instria vascularis marginal cells senescence model with mtDNA4834common deletion.Methods: We set up four experimental groups, as follows: group C: control group in whichMCs were not treated with D-galactose; group G: untransfected but with12mg/mlD-galactose for120hr; group B: transfected with Ad-GFP virus before treatment with12mg/ml D-galactose for120hr; and group P: transfected with Ad-PGC-1α-GFP DNArecombinant adenovirus before treatment with12mg/ml D-galactose for120hr. Then, wedetected CD rates by RT-PCR and detected the protein expression levels of PGC-1α, NRF-1,Tfam, NF-κB, FOXO-1and SIRT-1by western blot. Meanwhile we test the positiverepresentation of SA-β-gal and TUNEL (terminal deoxynucleotidyl transferase(TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labeling) in those four groups.Results: Our data show that the level of CD increased significantly in groups G and Bcompared with group C (p <0.01) by detecting the CD rates. Meanwhile, the level of CDdecreased significantly in group P compared with group B (p <0.01). We also found thatthe protein expression levels of PGC-1α, NRF-1, Tfam, NF-κB, FOXO-1and SIRT-1increased in our cell senescence model. After gene transfection, the protein expression levelof PGC-1α increased in P group compared with C group. Furthermore, the overexpressionof PGC-1α by transfection largely increased the expression levels of NRF-1and Tfam andsignificantly decreased the expression level of NF-κB, FOXO-1and SIRT-1in the cellsenescence model. Through detecting SA-β-gal and TUNEL, the positive representation inMCs increased in groups G and B compared with group C, while decreased in group Pcompared with group B.Conclusion: PGC-1α had been had been successfully transfected and overexpressed in MCs. Overexpression PGC-1α might protect MCs in this senescence model from agingassociated with decreasing of CD accumulation. Through the relative transcription factors,it can relive the aging and apoptosis of MCs.