C57BL/6鼠内耳形态发育及相关蛋白表达的比较研究/Comparing Study on Proteins Expressed During Morpholo

2018-10-23 12:07:53

表达 inner C57BL 毛细胞 D12



目的 为了深入了解C57BL/6小鼠内耳形态发育的一般规律及内耳发育过程中相关蛋白的表达情况。
方法 本实验以C57BL/6小鼠为实验对象。对E8-Da的C57BL/6小鼠的内耳发育时间段的内耳标本进行冰冻切片处理。运用HE常规染色、三维重建技术、扫描电镜和基因蛋白表达免疫组织化学分析技术进行研究分析。
结果 ①HE常规染色大体观上了解了C57BL/6小鼠内耳形态发育E8-Da期间的一般规律并且获得了E13的三维立体图像;②获得了各种听功能密切相关的结构的成熟时间点,基底膜(D10)、鼓阶前庭阶(D4)、螺旋器隧道(D12)、内螺旋沟(D10)、盖膜(D10)、毛细胞(D12)、螺旋神经元(D12)和蜗轴纤维(D12-D14);③完成了前神经决定蛋白、神经分化蛋白、神经营养因子和神经元特异性蛋白等内耳发育相关的蛋白的表达时限表并详细观察了其在内耳各个部位表达随发育变化的特点;④Calbindin在E16以后的螺旋神经元没有表达,但是在scarpa神经元持续表达。而NCS1在两者中都有表达;⑤Calbindin在E19-D22期间的毛细胞表达,但是在成年没有表达,并且内毛细胞出现表达较外毛细胞早,而表达消失较外毛细胞晚。MyosinVIIa在D1-D7的毛细胞清晰表达,但是D12表达渐弱,尤其是外毛细胞。⑥在C57BL/6鼠内耳发育过程中血管纹处有GAP-43, Peripherin, Neurofilament, BDNF, NT3和TUJ-1的表达;⑦神经元干细胞的标记物Islet1的表达从E16螺旋神经元所在的位置有大量的细胞核表达开始,一直到D7表达开始减弱,到D12表达的方式变为颗粒样表达。
结论 ①听功能相关的重要结构的成熟时间,提示C57BL/6小鼠的耳蜗要发挥听觉功能应该在D12和D14之间。②内耳发育过程中位于螺旋器隧道下的管腔结构不是螺旋血管,该结构的作用未明,可能是一个与螺旋器发育密切相关的发育期结构。③螺旋神经元与scarpa神经元在钙离子相关的生命活动中存在差异。④Calbindin在毛细胞表达有时限性,可作为新生毛细胞的特异性标志物。⑤ TUJ -1对内外毛细胞的传入和传出神经纤维染色,并推测外毛细胞中传入纤维发育比传出神经纤维迟。Peripherin对传入神经纤维染色而neurofilament可能对传出神经纤维染色。⑥血管纹中存在神经来源的细胞组织,可能是黑素细胞(暗细胞)。⑦在C57BL/6小鼠的内耳中,D7前的螺旋神经元仍然具有神经元干细胞的特性。⑧ TUJ-1在鸟类和C57BL/6鼠内外毛细胞中表达的差异,可能暗示着自然状态下可再生与不可再生毛细胞可以用TUJ-1染色来区分,但需进一步验证。



Purposes In order to make a good understand of the general principle in the morphological development of C57BL/6 mouse inner ear and of the expressions of proteins which were associated with the inner ear development.
Methods Take C57BL/6 mouse as experimental animals. The inner ear specimens From E8 to Da of C57BL/6 mouse were made into cryostat sections. Using HE staining, 3D reconstruction technology, SEM and immunohistochemi -strical methods to deal with the frozen sections.
Results ①Make clear of the general principal during the morphological development of the C57BL/6 mouse inner ear and reconstructed the 3D model of E13 inner ear;② The mature time of many histological structures associated closely with the hearing setout of C57BL/6 mouse were made clear as basilar membrane in D10,scala vestibular and tympanic D4,Corti’s tunnel D12,inner sulcus D10,tectorial membrane D10,hair cells D12,spiral ganglion neurons D12 and neurofilament in cochlear axis D12-D14.③ Outlined the time table and the patterns of the expressions of those proteins associated with the inner ear development which included proneural committed proteins, neuronal differential proteins, neurotrophic factors and neuron special proteins.④Calbindin was not expressed in spiral ganglion neurons after E16,but expressed continuously in scarpa ganglion neurons. However NCS1 can be found expressed in both of them. ⑤Calbindin could be labeled in hair cells between E19 and D22, but no expression was found in adult. And its expression first appeared and last disappeared in inner hair cells;The expression of MyosinVIIa could be found clearly between D1 and D7 and after that fading quickly especially in outer hair cells. ⑥many neurons special proteins and neurotrophin factor were expressed in the stria vascularis including GAP-43, Peripherin, Neurofilament, BDNF, NT3 and TUJ-1 during the development of the C57BL/6 mouse inner ear.⑦the neuron stem cell marker, Islet1 was expressed in the nuclear of the most spiral ganglion neurons from E16 to D7. After D7, the expression was fading;
Conclusions ① The mature time of the hearing function associated histological structures suggested that the hearing setout of the C57BL/6 mouse should between D12 and D14.②The vessel-like structure beneath the Corti’s tunnel during the inner ear development was not spiral vessel (SV).Until now, we can not ascertain the function of this structure and it maybe has some relationship with the development of the corti’s organ.③ scarpa ganglion neurons were different from spiral ganglion neurons in the biological activity involved in the Ca2+.④ The time limited expression model of Calbindin in hair cells show us its ability to work as the specific regeneration hair cells marker . ⑤ TUJ-1 were expressed in both afferent and efferent nerve fibers and maybe the afferent nerve fibers innervated out hair cells developed further than efferent ones. The Periphein was stained in afferent never fibers and neurofilament200KD in efferent nerve fibers.⑥the neuronal original tissue in the stria vascularis maybe melanin cells.⑦ The spiral ganglion neurons still had the characteristics of neuron stem cells before D7 in C57BL/6 mouse inner ear.⑧ the difference expression of TUJ-1 in the hair cells between avian and C57BL/6 mouse suggested that in normal conditions regenerable and unregenreable hair cells could be distinguished by TUJ-1 staining.