| Part Ⅰ The establishment of the senescenced marginal cell model with mtDNA4834bp common deletion induced by D-galactoseObject:To obtain an accessible cell model of presen(?)nce with significant mtDNA common deletion in the inner ear of rats.Method:The stria vascularis was dissected from neor(?)l Wistar rats (1-3days old), digested to cell suspension and inoculated in culture plates. The marginal cells (MCs) were identified by immunofluorescence of anti-cytokeratin (CK-18). MCs were treated with EpiCM contained gradient D-galactose (D-Gal)(0,2,4,6,8,10,12,14and16g/1) and D-Mannitol for3,7and11days, and then the activity of cells was detected by CCK-8. The proportion of the mitochondrial common deletion (CD) was detected with a TaqMan real-time PCR assay. MCs were cultured for1,5and11days in the control groups and the experimental groups treated with EpiCM contained12g/1D-Gal. We stained cells with Beta-Galactosidase Staining Kit to observe the senescence of cells. The apoptotic cells could be stained in situ by TUNEL.Result:The CK-18-specific-immunofuorescence was observed in the cytoplasm of MCs. The activity of MCs after gradient-concentration of D-Gal and D-Man treated was concentration-dependent declining,14g/1and16g/1had significant difference with the control group (P＜0.01). We selected12g/1to incubate MCs for different days (1,3,5,7,9,11,13and15days), the mtDNA4834bp CD represented concentration-and time-dependent accumulation and day5,7,9and11all had significant difference compared with control groups. Beta-galactosidase positive and TUNEL positive cells were found in MCs treated with12g/l D-Gal.Conclusion:D-galactose could successfully induce mtDNA4834bp common deletion in marginal cells and CD represented concentration-and time-dependent accumulation. The activity of MCs induced by D-Gal was ascribed to the changing of permeate pressure. We could observe significant senescence in MCs. Treated longer time, we could observe the apoptosis in MCs. We successfully established the senescenced marginal cell model with mtDNA4834bp common deletion induced by D-galactose. Part Ⅱ Functional status of Tom40and the importing of mitochondrial DNA polymerase y in the senescenced marginal cells induced by D-galactoseObject: To investigate the relationship between the changing status of Tom40importing the preproteins to mi(?)hondria and the importing of DNA polymerase y (DNA pol γ) in the accumulation of mt(?)A4834bp common deletion, and to explore the contribution of function of TOM comp(?)ex in the pathogenesis of age-related hearing loss.Method:Cochleae Were dissected from normal adult rats, made into5μm section and detected the expression of Tom40in the cochleae using immunofluorescence. After treated by12g/l D-Gal for11days, the expression of Tom40was detected by immunofluorescence. MCs were incubated in EpiCM contained gradient D-galactose (D-Gal)(0,2,4,6,8,10and12g/1) and EpiCM contained12g/l D-Gal for0,1,3,5,7,9and11days, extracted the total cell protein and the mitochondrial protein respectively. Western blot was used to analyze the expression of cellular and mitochondrial Tom40and DNA pol y.Result:(1) Tom40was expressed in several cell types in the stria vascularis, spiral ganglion and the organ of Corti in cochleae of rats.(2) The cultured marginal cells were rich in Tom40protein, which was especially localized in the cell nucleus and mitochondria, the expression of Tom40induced by D-Gal was significantly decreasing in the cytoplasm.(3) The expression of cellular Tom40was increasing firstly and then declining induced by the gradient D-Gal for3and7days, or12g/l D-Gal for different days. Therefore, the expression of the mitochondrial Tom40was enhanced.(4) The expression of cellular and mitochondrial DNA pol y was increasing firstly and then declining.Conclusion:The abundance of Tom40expressed in the cochlea and primary cultured marginal cells of rats, the changed function of Tom40could affect the normal function of cells to result in the hearing disease. The changing of Tom40and DNA pol y protein expression induced by D-Gal could reflect the mechanism of compensation and decompensation of cells. The consistence of changing of cellular Tom40and DNA pol y in the senescenced marginal cells suggested that Tom40could connect with the DNA pol y mitochondrial importing. The difference between the mitochondrial Tom40and DNA pol y suggested that other factors could participate in the process of mtDNA CD accumulation. The delayed changing of Tom40protein level in mitochondrion suggested the possible ex(?)ting of post-transcriptional modification. Part III Functional status of Tom20, Tom70and the mechanism of mtDNA common deletion accumulationObject:To further explore the changing status of Tom20and Tom70as the preproteins recognition sites in the accumulation of mtDNA CD, and to investigate the contribution of preprotein recognition before importing into mitochondria.Method:The expression of Tom20and Tom70was detected by imunofluorescence. MCs were incubated in EpiCM contained gradient D-galactose (D-(?)1)(0,2,4,6,8,10and12g/1) and EpiCM contained12g/l D-Gal for0,1,3,5,7,9and (?)1days, extracted the total cell protein and the mitochondrial protein respectively, extracted the total cell protein and the mitochondrial protein respectively. Western blot was used to analyze the expression of cellular and mitochondrial Tom20and Tom70.Result:The cultured marginal cells were rich in Tom20and Tom70protein, which was especially localized in the cytoplasm. The expression of cellular Tom20was decreasing in the accumulation of mtDNA CD; The expression of cellular and mitochondrial Tom70was increasing firstly and then declining in the accumulation of mtDNA CD.Conclusion:The abundance of Tom20and Tom70expressed iii primary cultured marginal cells of rats. The recognition activity of Tom20and Tom70to the mitochondrial preproteins could be more important to the importing of DNA pol y, and Tom20and Tom70could be more sensitive to the lower concentration of ROS induced by D-Gal. |
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