间充质对小鼠听觉上皮发育的影响

发布时间：2018-03-21 16:30

本文选题：内耳　切入点：间充质　出处：《皖南医学院》2015年硕士论文　论文类型：学位论文

【摘要】：目的:通过建立胚胎期小鼠耳蜗感觉上皮的体外培养模型,研究耳周间充质对耳蜗感觉前体细胞增殖和分化的影响,探讨其可能机制。方法:本实验采用胚胎期12.5天(E12.5)和13.5天(E13.5)的孕鼠,取其腹中胚胎鼠,在显微镜下取出完整的耳蜗感觉上皮,进行原代培养。对照组:带有相邻间充质的耳蜗感觉上皮;实验照组:不带间充质的单纯耳蜗感觉上皮。1).培养E12.5+6DIV或E13.5+5DIV,培养过程中采用倒置显微镜下观察听觉上皮的形态及生长状态,通过Myosin 7a标记毛细胞并对毛细胞进行计数,比较在有、无间充质的作用下,E12.5和E13.5的耳蜗感觉上皮的生长发育情况。2).采用RT-PCR技术测定Sox2和Math1基因的表达量。实验组和对照组的听觉上皮分别体外培养24h和48h,测定在不同培养阶段实验组和对照组耳蜗感觉上皮中Sox2及Math1基因的表达差异。3).在E12.5+6 DIV培养过程中加入6μg/ml BrdU,以BrdU标记增殖细胞,Myosin 7a标记毛细胞,定量研究间充质对耳蜗感觉前体细胞增殖和分化的影响。结果:1.实验组(去间充质组)中,E12.5和E13.5听觉上皮分别培养6天和5天,其整体形态和细胞排列较对照组(保留间充质组)均有一定差异。对照组E12.5天听觉上皮在体外无血清培养过程中,形态发育良好,细胞排列呈一定规律性,细胞间的连接较为紧密,呈相对规则的弧形。内毛细胞层和外毛细胞层之间的见有间隙,内毛细胞为1-2层,外毛细胞为4-6层,内毛细胞层相对于外毛细胞层较为紊乱。实验组E12.5听觉上皮生长缓慢,细胞间排列明显紊乱,无规则,细胞形态有一定的差异。毛细胞计数示:对照组723.75±79.54,实验组为515.35±45.63,两组有显著统计学(P0.05)。E13.5对照组听觉上皮内层毛细胞排列1-2层,排列整齐而紧密,外毛细胞也具有一定的规律性排列,尤其最外1-2层外毛细胞形态清晰可见,排列规整,实验组E13.5耳蜗感觉上皮毛细胞排列紊乱,连接较为疏松,内层毛细胞和外层毛细胞之间的界限不清晰,毛细胞计数示:对照组为1216.67±183.47.实验组为1097.15±204.67,实验组和对照组毛细胞计数虽无明显统计学差异,但实验组整体细胞数低于对照组。2.E12.5+24h听觉上皮中,对照组Math1基因表达和实验组组之间无统计学差异(P0.05);E13.5+24h、E12.5+48h和E13.5+48h听觉上皮中,对照组Math1基因表达高于实验组,实验组和对照组有统计学差异(P0.05)。E12.5+24h、E13.5+24h、E12.5+48h和E13.5+48h听觉上皮中,对照组Sox2基因表达高于实验组,实验组和对照组有统计学差异(P0.05)。3.E12.5听觉上皮体外培养6天后,BrdU+Myosin 7a双标阳性细胞:对照组18.9±2.77;实验组:13.7±2.91。实验组和对照组有统计学差异(P0.05)。结论:E12.5耳周间充质能促进耳蜗感觉前体细胞增殖,但更关键的是促进耳蜗感觉前体细胞向毛细胞分化。胚胎鼠听觉上皮形态发生过程中,耳周间充质促进Math1和Sox2基因表达上调。
[Abstract]:Objective: to study the effect of periauricular mesenchyma on the proliferation and differentiation of sensory precursor cells in mouse cochlea in vitro by establishing an in vitro culture model of mouse cochlear sensory epithelium. Methods: the pregnant mice of 12.5 and 13.5 days of embryonic stage were used in this study. The embryonic mice in their abdomen were taken out and the complete sensory epithelium of cochlea was removed under microscope. Primary culture. Control group: cochlear sensory epithelium with adjacent mesenchymal cells; In the experimental group, the sensory epithelium of cochlea was cultured without mesenchyma. The morphology and growth state of auditory epithelium were observed under inverted microscope. Hair cells were labeled with Myosin 7a and the hair cells were counted. There are more, The growth and development of cochlear sensory epithelium of E12.5 and E13.5 under the action of non-mesenchymal cells. The expression of Sox2 and Math1 genes was measured by RT-PCR technique. The auditory epithelium of the experimental group and the control group were cultured in vitro for 24 hours and 48 hours, respectively. The expression of Sox2 and Math1 genes in the sensory epithelium of the cochlea of the experimental group and the control group was different from that in the control group. 6 渭 g / ml BrdU was added into the culture process of E12.56 DIV, and the hair cells were labeled with BrdU labeled with Myosin 7a. The effects of mesenchymal cells on the proliferation and differentiation of cochlear sensory precursor cells were quantitatively studied. Results 1. In the experimental group (demesenchymal group), the auditory epithelium of E12.5 and E13.5 were cultured for 6 days and 5 days, respectively. The morphology and cell arrangement of the E12.5 day auditory epithelium in the control group were different from those in the control group (retention of mesenchymal cells), and the morphology of the auditory epithelium in the control group was well developed during the serum-free culture in vitro, and the cell arrangement was regular. There was a gap between the inner hair cell layer and the outer hair cell layer. The inner hair cell had 1-2 layers, and the outer hair cell had 4-6 layers. The inner hair cell layer was more disordered than the outer hair cell layer. In the experimental group, the auditory epithelium of E12.5 grew slowly, the intercellular arrangement was obviously disordered, and there was no rule. The number of hair cells was 723.75 卤79.54 in the control group and 515.35 卤45.63 in the experimental group. Especially, the outermost 1-2 layers of outer hair cells were clearly visible and arranged regularly. In the experimental group, the sensory hair cells of the E13.5 cochlea were arranged in disorder, the connection was looser, and the boundary between the inner hair cells and the outer hair cells was not clear. The number of hair cells in the control group was 1216.67 卤183.47.The number of hair cells in the experimental group was 1097.15 卤204.67. Although there was no significant difference between the experimental group and the control group, the total number of hair cells in the experimental group was lower than that in the control group. There was no significant difference between the expression of Math1 gene in the control group and that in the experimental group. The expression of Math1 gene in the auditory epithelium of the control group was higher than that in the experimental group. The expression of Math1 gene in the control group was higher than that in the experimental group. There was a significant difference between the experimental group and the control group. Sox2 gene expression in control group was higher than that in experimental group. There were significant differences between the experimental group and the control group (P 0.05N. 3.E12.5 auditory epithelium cultured in vitro for 6 days): BrdU Myosin 7a double labeled positive cells: control group 18.9 卤2.77; experimental group 13.7 卤2.91.There was significant difference between the experimental group and the control group (P0.050.Conclusion: 1. 05% E 12.5 ear filling can promote cochlea. Sensory precursor cell proliferation, However, it is more important to promote the differentiation of cochlear sensory precursor cells into hair cells. During the morphogenesis of auditory epithelium in embryonic mice, mesenchymal mesenchyma promotes the up-regulation of Math1 and Sox2 gene expression.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R764.43

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