Expression Of α₁ Na, K-ATPase And PDCD5 In The Cochlea Of The C57BL/6J Mouse At Different Ages And Relationship With AHL

Part I Establishment of marginal cell culture from stria vascularis of adult mouse and the expression of KCNQ1 in MCObjective:To provide an in vitro cell model for further study of the function of marginal cell(MC), primary cultures of marginal cells of adult mouse were established.Methods:The marginal cells were isolated from the stria vascularis of adult mice by CollagenaseⅡto culture. The morphologic changes of the cells were observed under an inverted lightmicroscope and the growth curve of the cell was determined by MTT assay. The ultrastructure of the cultured cells was observed by transmission electron microscopy. The expression of CK18 and KCNQ1 were detected by immunofluorescence and reverse transcription- polymerase chain reaction (RT-PCR).Results:The primary cultured cells attached within 24-48 h of seeding and proliferated in an island-like monolayer. After one week, more and more epithelial-like cells had a tendency to become confluent and became tightly packed, growing into a monocellular layer,"cobblestone-like appearance. Transmission electron microscope showed typical epithelia with microvilli on the cells surface, massive mitochondria and rough endoplasmic reticulum in the cells. Expression of CK18 and KCNQ1 in MC was confirmed by immunofluorescence staining, and expression of CK18 mRNA and KCNQ1 mRNA was also detected by RT-PCR.Conclusions:The cell culture system of marginal cells of adult mouse has been successfully established by enzyme digestion which can provide a stable source of MC for investigation of the ongoing function of the marginal cell.PartⅡExpression ofα1Na,K-ATPase in the cochlea of the C57BL/6J mouse at different ages and relationship with presbycusisObjective:To investigate the age related changes of the expression ofα1Na,K-ATPase in the cochlea of the C57BL/6 (C57) mice, and also discusses the relationship ofα1Na,K-ATPase and Presbycusis.Method:Auditory function of C57 mice t different ages between 3 and 12 months old was measured by auditory brainstem response (ABR) respectively. The changes ofα1Na, K-ATPase protein in the cochlea was detected by immunofluorescence and Western blot, the expression ofα1Na, K-ATPase mRNA and caspase-3 mRNA were detected using RT-PCR.Result:The average ABR threshold value from 6-month-old C57 mice was not significantly different from that of 3-month-old mice (P>0.05), but the difference between the average ABR threshold value from mice of 12 months old than 9 months and that of 3-month-old mice was highly significant (P<0.01), and the average value appeared to progressively increase with age. The localization ofα1Na, K-ATPase was found mainly in the stria vascularis and the spiral ligament, and the expression ofα1Na, K-ATPase protein was decrease in the stria vascularis and the spiral ligament with age. The change ofα1Na, K-ATPase mRNA expression was in accordance with that ofα1Na,K-ATPase protein expression by the real-time PCR.Conclusion:Expression ofα1Na, K-ATPase in the cochlea of C57 mice gradually decreased with increasing age, and might play an important role in pathogenic mechanism of Age-related hearing loss (AHL).PartⅢExpression and significance ofα1Na, K-ATPase in the marginal cell from stria vascularis of adult C57BL/6J miceObjective:To study the expression and significance of ofα1Na, K-ATPase in the marginal cell from stria vascularis of adult C57BL/6J (C57) mice, and provides an experimental basis for explored K+ ion recycling.Method:The marginal cell isolated by CollagenaseⅡto culture. The expression ofα1Na, K-ATPase ion transporters in the marginal cell of C57 mice was detected by immunofluorescence and the expression ofα1Na, K-ATPase by RT-PCRResult:Theα1Na, K-ATPase were cultured and purified in vitro. The immunofluorescence results showedα1Na, K-ATPase only expressed in membrane of marginal cell, and the mRNA expression ofα1Na, K-ATPase was also detected by RT-PCR.Conclusion:The present results suggest that theα1Na,K-ATPase expression in membrane of of marginal cell may play an important role in the K+ ion recycling of the inner ear.Part IV Expression of PDCD5 and caspase-3 in the cochlea of different age C57BL/6J mice and relationship with presbycusisObjective:To investigate the age related changes of the expression of PDCD5 and caspase-3 in the cochlea of the C57BL/6(C57)mice. This paper also discusses the relationship of PDCD5 and caspase-3 and the possible role in the pathogenesis of Presbycusis.Method:C57 mice of 3,6,9 and 12 months old were selected and divided into 4 groups (fifteen mice each). Auditory function of C57 mice was measured by auditory brainstem response (ABR) respectively. The changes of PDCD5 and caspase-3 protein in the cochlea was detected by immunohistochemistry and Western blot, the changes of PDCD5 mRNA and caspase-3 mRNA were detected using RT-PCR.Result:With an increase of age,The mean value for ABR thresholds in response to click,4 kHz and 8 kHz sound stimulus of C57 mice gradually increased, the expression of PDCD5 and caspase-3 was increased also. At 3 months and 6 months of age in the cochlea of C57, all sorts of expression of PDCD5 and caspase-3 and the expression was enhanced with age. There was an evident expression at 9 months of age, but the highest expression was detected at 12 months of age. The PDCD5 and caspase-3 expression was statistically different in each group (P<0.05).The change of PDCD5 and caspase-3 mRNA expression was in accordance with that of PDCD5 and caspase-3 protein expression by the real-time PCR.Conclusion:The increased expression levels of PDCD5 and caspase-3 in the cochlea of C57 mice with age, the result s suggested that the expression of PDCD5 and caspase-3 participated in the regulatory procedure of apoptosis and might play an important role in pathogenic mechanism of Presbycusis.