Effects Of Mesenchyme On Development Of Auditory Epithelium In Mice

Objective:To establish a practical method of auditory epithelia culture model in vitro for embryonic mice and study the effect of mesenchyme on the differentiation and proliferation of the mouse auditory epithelium and the expression of Sox2 and Math1 in the developing mouse auditory epithelium.Method: This experiment used the pregnant mouses with embryonic period of 12.5days(E12.5) and 13.5 days(E13.5).Then we taken the fetal mouses from them,from which we extracted complete auditory epithelium by microscopic operation.The experiment group was treated with thermolysin removing the jacent mesenchymal for primary tissue culture,control group did not do the treatment for primary culture.Two groups were cultured to E12.5+6d and E13.5+5d.Then,observate the growth state and cell morphology of auditory epithelial under the inverted microscope and count the number of hair cells by immunofluorescence.Analyze the difference between the experimental group and the control group.Use RT-PCR technology to determinate the amount of expression of Sox2 and Math1 while the experimental group and the control group were cultured to 24 h and 48 h.Measure the expression of Sox2 and Math1 at different culture stages in the auditory sensory epithelium.To research the efect of mesenchyme on the proliferation and differentiation of precursor cells by marking Myosin7 a and Br DU.RESULT:1.Immunofluorescence:in the experimental group(without mesenchymal tissue),epithelial tissue of E12.5 and E13.5 cultured for 6 days and 5 days had significant differences with the control group(with mesenchymal tissue) in growth state and cell morphology. Epithelial tissue of E12.5 in control group developed well withigorous growth,regular morpholog and closely connected cells.There was gap between the inner hair cell layers and the outer hair celllayers.Inner hair cells with 1-2 layers is more disorganized than outer ones with 4-6 layers.Epithelial tissue of E12.5 in experimental group growed showly and were arranged in disorder.Hair cells were irregular without significant morphology.Hair cell number:the control group 723.75 ±79.54, the experimental group 515.35 ±45.63.The two groups had significant statistical difference(P<0.05).Inner hair cells of E13.5 in experimental group arranged neatly and closely in 1-2 layers.The outer hair cells also has certain regularity of arrangement, especially the cells in outer 1-2 layers had obvious morphology.Hair cells of E13.5 in experimental group arranged in disorder and connection were loose.The boundary between the inner hair cells and outer hair cells was not clear.Hair cell number count:The control group 1216.67 ±183.47,experimental group 1097.15 ±204.67. There is no significant statistical difference between experimental group and control group in hair cell count, but the cell number of experimental group as a whole was still lower than the one in control group.2.RT-PCR:In E12.5 + 24 h, there was no significant statistical difference between control group and experimental in the expression of Math1 gene(P > 0.05). In E13.5+24h、E12.5 + 48 h and E13.5 + 48 h,the expression of Math1 gene in control groups was higher than the one in experimental group, there was statistical difference(P < 0.05).In E12.5 + 24h、E12.5+48h、E13.5+24h and E13.5+48h,the expression of Sox2 in the expression of the experimental groups was lower than the control groups, with statistical difference(P < 0.05).3.By marking Br DU and Myosin7 a of auditory epithelium in E12.5+6d,the result showed :18.9±2.77 in the control group; The experimental group: 13.7 ±2.91. The experimental group and the control group had statistically difference(P < 0.05).Conclusion:The adjacent mesenchymel promotes differentiation and development ofauditory epithelial,The mesenchyme contributes to upregulate the expression of Math1 and Sox2 in developing auditory epithelial.