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目的检测Smad4基因在成年小鼠耳蜗中是否有表达及表达部位的分布情况,以进一步研究Smad4基因在内耳发育以及听功能调控上的作用。方法动物由军事医学科学院发育和疾病遗传学研究室杨晓研究员提供,动物获取是在C57BL/6J和Blackswiss两种遗传背景的小鼠应用Cre-LoxP系统进行条件基因打靶,于小鼠胚胎早期在软骨细胞内将Smad4基因剔除。并生成同窝三种基因型小鼠—野生型Smad4+/+,杂合子Smad4+/-,纯合子Smad4-/-用于本研究。对同窝三种不同基因型小鼠的耳蜗冰冻切片应用免疫组织化学方法染色观察,阴性对照为野生型小鼠耳蜗的冰冻切片并用PBS代替一抗,阳性对照为小鼠肾脏组织冰冻切片。结果Smad4在三种基因型小鼠耳蜗均有广泛表达,表达部位主要集中于血管纹、螺旋韧带、基底膜、盖膜、毛细胞、支持细胞、螺旋神经节细胞等处,其中血管纹和基底膜表达最为明显。Smad4在野生型和杂合型小鼠的耳蜗内表达情况基本一致,但纯合子小鼠耳蜗内表达与前两者相比为较低的水平,但未完全消失。结论Smad4在正常小鼠的耳蜗广泛存在,该基因是耳蜗中广泛表达的蛋白中的一种。作为Bmp4信号通路下游信号因子,它可能在骨性耳蜗的形成、内耳感觉细胞的分化成熟过程中起着重要的作用。
Objective To detect whether Smad4 gene is expressed in the cochlea of adult mice and the distribution of Smad4 gene in order to further study the role of Smad4 gene in inner ear development and hearing function regulation. Methods The animals were provided by Yang Xiao, a researcher of Development and Disease Genetics Laboratory, Academy of Military Medical Sciences. Animals were obtained by using the Cre-LoxP system for conditional gene targeting in mice of both C57BL / 6J and Blackswiss genetic backgrounds. Smad4 gene is deleted in chondrocytes. Three genotypes of littermates - wild-type Smad4 + / +, heterozygous Smad4 +/- and homozygous Smad4 - / - were used in this study. The cochlear frozen sections of three different genotypes of the same litter were stained by immunohistochemical staining. The negative control was frozen sections of the wild-type mice cochlea and PBS was used instead of the primary antibody. The positive control was frozen sections of the kidney of mice. Results Smad4 was widely expressed in the cochlea of three genotypes mice. The expression sites were mainly located in vascular lines, spiral ligaments, basement membrane, capillaries, hair cells, supporting cells and spiral ganglion cells. Membrane expression of the most obvious. The expression of Smad4 in the cochlea of wild-type and heterozygous mice was basically the same, but the homozygous mouse cochlear expression was lower than the former two, but did not disappear completely. Conclusion Smad4 is widespread in the cochlea of normal mice and is one of the most widely expressed proteins in the cochlea. As a downstream signal factor of Bmp4 signaling pathway, it may play an important role in the formation of bony cochlea and the process of differentiation and maturation of inner ear sensory cells.