小鼠听觉上皮发育及其相关分子机制

发布时间：2018-03-25 23:25

本文选题：小鼠　切入点：听觉　出处：《复旦大学》2010年博士论文

【摘要】：上皮和间充质组织之间的信号交流和相互作用对器官的发生和发育起重要作用。BMP4是TGFβ超家族成员中的一个亚型,BMP4及其拮抗因子Noggin调控听觉上皮和内耳周围间充质的发生及发育。然而,BMP4在内耳感觉上皮发育中的作用目前存在较大的争议。我们首先采用胚胎期小鼠第11.5天(E11.5)～13.5天(E13.5)的听觉上皮体外培养,建立胚胎期小鼠听觉上皮的体外培养模型。所有组织的培养时间均等同于体内发育至E18.5的时间(即E11.5+7 d in vitr(DIV), E12.5+6 DIV,E13.5+5 DIV),观察听觉上皮形态和毛细胞分化情况。结果表明听觉上皮可以在体外无血清培养环境下存活并生长,其中E12.5～E13.5的听觉上皮培养后发育形态及毛细胞分化接近正常发育形态,是研究毛细胞发育机制的合适模型。采用小鼠听觉上皮体外无血清培养模型,以thermolysin获得纯听觉上皮作为实验组,带有相邻间充质的听觉上皮为对照组,研究相邻间充质对听觉上皮发育的影响。结果显示：各胚胎期小鼠听觉上皮培养后毛细胞的数量,对照组均高于实验组。E11.5+7 DIV对照组为114.43±17.92,实验组为66.78±20.64,两组统计学差异有显著性(P0.01)；E12.5+6 DIV对照组为519.75±98.11,实验组为299.71±23.91,两组统计学差异有显著性(P0.01)；E13.5+5DIV对照组为1214.38±231.88,实验组为1158.67±176.09,两组统计学无差异。其中,在E12.5和E13.5实验组内毛细胞发育不良,而E12.5对照组内毛细胞呈1-3排不规则排列,E13.5对照组的内毛细胞和外毛细胞出现明显的界限,内毛细胞呈纵向单排生长。另外,对照组的毛细胞/支持细胞比例也均高于实验组。以上结果表明小鼠听觉上皮相邻的间充质组织对耳蜗感觉上早期形态发育及毛细胞分化有促进作用。我们采用E11.5～E13.5小鼠听觉上皮体外无血清培养模型,通过加入外源性BMP4蛋白或其抑制剂Noggin,研究BMP4在听觉上皮发育中的作用。加入外源性BMP4后,实验组的毛细胞数量均高于对照组,并呈剂量依赖性。E11.5+7 DIV对照组毛细胞数量(114.43±17.915)和bmp lOng组(150.80±29.107)有统计学差异(P0.05),和bmp 20ng组(198.33±40.741)有统计学显著差异(P0.01),E12.5+6 DIV对照组毛细胞数量(519.75±98.112)和bmp 20ng组(650.29±173.241)有统计学差异(P0.05),但与bmp 10ng组(588.50±111.524)却无统计学差异。E13.5培养后实验组虽然和对照组相比均无统计学差异,但在统计数量上均有增多。另外,各实验组的毛细胞／支持细胞比例也均高于对照组,但它们的作用与开始培养的发育时期和剂量相关,E11.5+7 DIV实验组(bmp 10ng组：0.3670±0.0616；bmp 20ng组：0.4386±0.1185)虽然在统计数值上有增高,但和对照组(0.3267±0.0755)却无统计学差异。E12.5+6 DIV对照组(0.6657±0.0214)和bmp 10ng组(0.7038±0.0407)有统计学差异(P0.05),而和bmp 20ng组(0.7641±0.04521)有统计学显著差异(P0.01),而E13.5+5DIV对照组(0.6820±0.0187)和bmp 20ng组(0.7944±0.0806)有统计学差异(P0.05),但和bmp 10ng组(0.7064±0.0967)却无统计学差异。通过在培养中加入Brdu标记增殖细胞,发现BMP4组通过增殖而来的毛细胞数量和对照组相比较并无差别。Shh和Sox2是听觉器官发育,特别是前感觉区(prosensory domain)形成的关键基因。我们发现加入外源性BMP4后,Sox2、Shh基因的表达明显下调,E12.5+6 DIV bmp4 20ng组的Sox2 mRNA的量为对照组的81.2%,而Shh mRNA的量仅为对照组的43.7%。当加入BMP4抑制齐(?)0.3μg/ml Noggin后,导致毛细胞显著减少(E11.5+7 DIV：40.60±14.188；E12.5+6 DIV：65.14±22.974；E13.5+5 DIV：231.17±90.061)均远少于对照组,统计学差异有显著性(P0.01)。这些结果表明：听觉上皮发育早期,Bmp4能促进感觉前体细胞分化为毛细胞,其作用可能与抑制(?)Sox2、Shh基因表达有关。另外,采用免疫荧光共标技术,观察Pax2、Sox2及Proxl在小鼠内耳发育中的时空表达模式及相互关系。结果显示：在E9.5,Pax2主要分布在听泡的中间和内侧,随着前感觉区的形成,Pax2在前感觉区表达逐渐下调；当原始的毛细胞开始表达myosin7a时,它重新选择性上调表达于毛细胞中并维持至出生后第7天；而在前庭毛细胞中,至出生后第18天Pax2仍有表达。Sox2具有与Pax2明显不同的表达方式,开始主要分布在听泡背外侧,随着前感觉区的形成,逐渐局限在感觉前体细胞,随后在未成熟的毛细胞和支持细胞中表达；毛细胞逐渐分化成熟时,在耳蜗毛细胞表达逐渐下调,最后仅仅局限在支持细胞,而前庭的毛细胞和支持细胞中在出生后18天仍持续表达Sox2。Prox1表达在原始的毛细胞和未成熟的支持细胞,表达时间明显比Sox2短。在耳蜗,Proxl仅仅短暂地表达在原始外毛细胞和发育中的Deiter细胞和Pillar细胞,在出生后18天时已无Prox1表达；而在前庭,出生时就无Proxl阳性表达。转录因子Pax2、Sox2及Prox1在小鼠内耳发育中互相联系又有不同的时空分布特点,表明它们在内耳发育中具有不同的功能,特别是它们在前庭和耳蜗不同的表达模式表明前庭和耳蜗感觉细胞的分化形成可能具有不同的分子机制。
[Abstract]:Signal exchange and interaction between epithelial and mesenchymal tissues of organs and plays an important role in the development of.BMP4 is a subtype of TGF beta super family members, around BMP4 and its antagonist Noggin regulation of auditory epithelium and inner ear between mesenchymal and development. However, the role of BMP4 in developing auditory epithelium in the current controversial.
We used mouse embryonic day 11.5 (E11.5) to 13.5 days (E13.5) cultured in vitro cultured auditory epithelial model, the establishment of embryonic auditory epithelium in vitro. The incubation time all tissues were equivalent to E18.5 in vivo development time (E11.5+7 (DIV) d in vitr, E12.5+6 DIV, E13.5+5 DIV), observation of auditory hair cells morphology and epithelial differentiation. The results show that the auditory epithelium can be cultured in serum-free medium in vitro under the environment of the survival and growth of the E12.5 ~ E13.5 after cultured auditory epithelial morphogenesis and differentiation of hair cells close to the normal development of form, is the appropriate model to study the mechanism of hair cell development.
Mouse auditory epithelium in vitro serum-free culture model by thermolysin to obtain the pure auditory epithelia as the experimental group, with the adjacent mesenchymal auditory epithelium as control group. The effect of mesenchymal adjacent to auditory epithelial development. The results showed that the number of embryonic auditory hair cells after cultured epithelium, the control group were higher than that of the experimental group.E11.5+7 DIV control group is 114.43 + 17.92, 20.64 + 66.78 in the experimental group, the two groups had significant statistical difference (P0.01); E12.5+6 DIV group is 519.75 + 98.11, 23.91 + 299.71 in the experimental group, the two groups had significant statistical difference (P0.01); E13.5+5DIV control group is 1214.38 + 231.88. The experiment group is 1158.67 + 176.09, no statistical difference between the two groups. Among them, E12.5 and E13.5 in the experimental group in hair cell dysplasia, E12.5 group of inner hair cells are arranged in 1-3 rows of irregular arrangement, E13.5 control group of inner hair cells And the outer hair cells appear obvious boundaries, a longitudinal single row of inner hair cell growth. In addition, the control group of hair cells / Sertoli cell ratio were also higher than the experimental group. The above results indicated that mouse auditory epithelial mesenchymal tissue adjacent to cochlear sensory hair cells on early morphogenesis and differentiation have stimulative effect.
We use E11.5 to E13.5 mouse auditory epithelium in vitro serum-free culture model by adding exogenous BMP4 protein or its inhibitor Noggin, study the role of BMP4 in auditory epithelial development. After addition of exogenous BMP4, the number of hair cells in experimental group were higher than those in the control group, and showed a dose dependent.E11.5+7 DIV number in the control group. Cells (114.43 + 17.915) and BMP lOng group (150.80 + 29.107) there were significant differences (P0.05, BMP) and group 20ng (198.33 + 40.741) was statistically significant difference (P0.01), E12.5+6 DIV hair cell number in the control group (519.75 + 98.112) and BMP 20ng group (650.29 + 173.241) there were significant differences (P0.05), but with the BMP 10NG group (588.50 + 111.524) but there was no significant difference in.E13.5 culture after the experimental group and the control group while there was no significant difference in the number of statistics, but has increased. In addition, the hair cell / support proportion of cells in each experimental group It is higher than the control group, but their role and begin to develop period of training and dose related, E11.5+7 DIV group (BMP 10NG group: 0.3670 + 0.0616 BMP; group 20ng: 0.4386 + 0.1185) although the statistical numerical has increased, but the control group and (0.3267 + 0.0755) but no statistically significant difference.E12.5+6 DIV group (0.6657 + 0.0214) and BMP 10NG group (0.7038 + 0.0407) there were significant differences (P0.05), and BMP and 20ng group (0.7641 + 0.04521) was statistically significant difference (P0.01, E13.5+5DIV) and control group (0.6820 + 0.0187) and BMP 20ng group (0.7944 + 0.0806) there were significant differences (P0.05), but BMP and 10NG group (0.7064 + 0.0967) but no significant difference. By adding the Brdu marker of proliferating cells in culture, proliferation and BMP4 group by the number of hair cells compared with the control group there is no difference between.Shh and Sox2 is the auditory organ development, especially the first sensory area (pros Ensory domain) key gene formation. We found that the addition of exogenous BMP4, Sox2, Shh gene expression was down regulated, 81.2% E12.5+6 DIV BMP4 20ng Sox2 mRNA group was the control group, while Shh mRNA is only 43.7%. of the control group when adding BMP4 inhibitor (?) 0.3 g/ml Noggin after the result of hair cells was significantly reduced (E11.5+7 DIV:40.60 + 14.188 E12.5+6 + 22.974 E13.5+5; DIV:65.14; DIV:231.17 + 90.061) are far less than the control group, there was significant difference (P0.01). These results suggest that the early development of the auditory epithelium, Bmp4 can promote the differentiation of progenitor cells into sensory hair cells, which may be related to inhibition of Sox2 (?), the expression of Shh gene.
In addition, by immunofluorescence labeled technique, observation of Pax2, expression in mouse inner ear development in Sox2 and Proxl and the relationship between them. The results showed that in E9.5, Pax2 mainly distributed in the middle and medial to the bubble, as before the formation of sensory area, the expression of Pax2 in the sensory area gradually reduced; when hair cells the original expression of myosin7a, it re expression in selective hair cells and maintained up to seventh days after birth; and in the vestibular hair cells, to eighteenth days after birth, Pax2 still has the expression of.Sox2 and Pax2 with different modes of expression, beginning mainly distributed in the dorsal lateral auditory vesicle, as before the formation of sensory area the feeling was gradually limited in precursor cells, then the expression in hair cells and supporting cells in immature hair cells; differentiation gradually mature, gradually down regulated expression in cochlear hair cells, finally only in support of the cells. Hair cells and supporting cells in court in 18 days after birth is still the expression of Sox2.Prox1 in the original hair cells and immature support cells, the expression time was significantly shorter than Sox2. In the cochlea, Proxl only briefly in the original expression of outer hair cells and Deiter cells and Pillar cells in the development of the expression after birth 18 the day has no Prox1; while in the vestibule, no positive expression of Proxl was born. The transcription factor Pax2, Sox2 and Prox1 in the mouse inner ear development in contact with each other and the temporal and spatial distribution characteristics of different, indicating that they have different functions in the inner ear development, especially in the vestibular and cochlear different expression patterns show the differentiation of sensory cells of the vestibular and cochlear formation may have different molecular mechanisms.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764

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