Smad4 基因在野生型和基因缺陷小鼠耳蜗内的定位和分布

目的检测Smad4基因任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学相比为较低的水平．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。

Location and distribution of the Smad4 gene in wild-type and mutant mice

Objective To investigate whether the Smad4 gene exists in the cochlea of postnatal mice and to detect its expression and distribution in the cochlea. Methods We prepared frozen sections of cochleae from three monse genotypes: Smad4 (+/+), Smad4 (+/-) and Smad4 (-/-). Smad4 expression of the cochlea was detected by immunohistochemical technique. As a negative control, frozen sections of the cochlea were obtained from wild-type mice with no antibodies and a positive control, frozen sections of mouse kidney were used. Results Smad4 genes were expressed throughout the postnatal cochlea of three mouse genotypes at a high level. The expression concentrated in the vascular stria, spiral ligament, basal membrane, tectorial membrane, hair cells, supporting cells and spiral ganglion cells. Expressions in the vascular stria and organ of Corti were stronger comparing to other inner ear structures. Conclusion The Smad4 gene is one of genes existing in the postnatal cochlea extensively. As a signalling factor at downstream of the Bmp4 pathway, the Smad4 gene may play an important role in the process of formation of the osseous otocyst and differentiation of sensory cells.