The Research On Effects Of Mecom On Proliferation And Differentiation Of Cochlear Supporting Cells

Objective: To observe the expression characteristics of Mecom gene at different stages in mice inner ear,and to discuss the influence of Mecom gene in the process of supportcells proliferation and differentiation in the development of the inner ear.Methods: 1.Select embryonic mouse ranging from E9.5 day to E18.5 day in C57 BL / 6 mice,using frozen serial section and immunofluorescence staining to observe the expression of Mecom in the inner ear;2.Up-regulation Mecom gene through the technology of adenovirus transfection in basilar membrane;3.Up-regulation the expression of Mecom genes and then to investigate it,s effort and mechanism in the process of proliferation and trans-differentiation of support-cells by immunofluorescence staining and q PCR experiment.Results: 1.The expression of Mecom protein in the development of inner ear:When embryonic development to E10.5 day,the expression Mecom gene started appeared around the sensory epithelium weakly;The expression Mecom gene was observed in sensory epithelium only at E12.5 day period.2.The Adenovirus transfection in basilar membrane :: The transfection efficiency of support-cells in apex of basilar membrane was 7.2 ±4%,50±3.3% in mid and 42±6.3% in base,The transfection efficiency of hair cell transfection was 30.7 ± 2.7% in apex and 50±3.3% in mid.Real-time fluorescent quantitative PCR and Western Blot experiment show that with the transfection of Ad-Mecom,the gene and protein expression of Mecom was up-regulation,P<0.01;3.The influence of the Mecom genes to support cells : After up-reglulation the expression of Mecom,we observed the proliferation of support cells at the top and middle of the basilar membrane,and the trans-differentiation of support cells was observed in middle of basilar membrane.Real-time fluorescent quantitative PCR show the expression of Axin2、 Sp5 was obviously increased,and gene transcription level of Hey1、Hes1 、Cdkn2b and Smad3 significantly decreased.Conclusion: The timeliness and regional of Evi-1 gene expression in the development period of inner ear was obvious,Evi-1 may have a significant function during the process of hair cells development.The timeliness and regional of Mecom was obvious.Cell proliferation could observed in support cells after up-regulation the Mecom gene,and we found the trans-differentiation of support cell.To sum up,after rising the expression of Mecom genes could induce the process of support-cell proliferation and trans-differentiation through Wnt 、Notch and TGF/BMP signaling pathways.