转化生长因子β1在大鼠胚胎耳蜗中的表达

doi:10.3969/j.issn.2095-4344.2013.46.004

摘要/Abstract

摘要：

背景：目前人们对耳蜗的发育做了大量的研究工作，但主要集中在病理情况及发育畸形上，而对正常胚胎耳蜗发育过程及相关因子表达的研究较少。
目的：观察转化生长因子β1在大鼠胚胎耳蜗中的表达情况。
方法：取SD妊娠大鼠56只，从E9-E19逐日将孕鼠断颈，剖腹，迅速取出胚胎，解剖显微镜下修剪内耳标本，经脱水、脱钙、定向石蜡包埋、切片处理。苏木精-伊红染色后光镜下观察内耳形态学演变过程。免疫组织化学SABC法观察转化生长因子β1在胚胎耳蜗的表达。
结果与结论：大鼠内耳的发育开始于E8的外胚层增厚区，E9就可观察到听泡及其周围间充质的出现，E9.5听囊及听骨链开始发育，E12.5蜗管始基形成，E18.5耳蜗管发育完成，E16 Corti’s器发育形成，直到出生后内耳的结构和功能才发育完全。转化生长因子β1在E12.5-E19期均有表达，表达情况由弱到强再减弱，E14.5时最强。转化生长因子β1在大鼠耳蜗上皮发育中的表达呈明显的阶段性及组织特异性分布的特点，说明转化生长因子β1可能参与大鼠耳蜗上皮的发育过程。

关键词: 组织构建, 组织构建与生物活性因子, 转化生长因子β1, 耳蜗, 发育, 胚胎, 大鼠

Abstract:

BACKGROUND: Many researches on the development of cochlea have been made, but mainly depend on the pathological conditions and developmental deformity, while the researches on the development process of normal embryonic cochlea and expression of related factors are rare.
OBJECTIVE: To investigate the expression of transforming growth factor β1 in rat embryonic cochlea.
METHODS: Fifty-six Sprague Dawley rats were selected and the rats were pregnancy, and the embryos were obtained from the rats, trimmed the inner ear specimens under the anatomical microscope, and then the specimens were treated with dehydration, decalcification, directly paraffin embedding and slicing processing. The morphology evolution of inner cochlea was observed under light microscope after hematoxylin-eosin staining. The expression of transforming growth factor β1 in rat embryonic cochlea was observed by immunohistochemical streptavidin biotin-peroxidase complex method.
RESULTS AND CONCLUSION: The development of rat inner ear began in ectoderm thickening area of E8 phase, otocyst and the emergency of the mesenchymal could be seen in E9 phase, otocyst and ossicular chain began to develop in E9.5 phase, formation of cochlear duct anlage could be seen in E12.5 phase, cochlea tube development was completed in E18.5 phase and Corti’s formation could be seen in E16 phase. The structure and function of the inner ear could be fully developed after birth. The expression of transforming growth factor β1 could be seen in E12.5-E19 phases, and the expression was changed from weak to strong and then weakened further, and strongest in E14.5 phase. This suggested that transforming growth factor β1 may be involved during the development of rat cochlear epithelium.

Key words: transforming growth factor beta, cochlea, embryonic development, rats, Sprague-Dawley

中图分类号:

R318

李翠娥，史艳莉. 转化生长因子β1在大鼠胚胎耳蜗中的表达[J]. 中国组织工程研究, 2013, 17(46): 8000-8003.

Li Cui-e, Shi Yan-li. Expression of transforming growth factor-beta 1 in rat embryohic cochlea[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(46): 8000-8003.

图/表（结果） 2

参考文献

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引言

材料方法

讨论

大鼠是胚胎发育研究领域被广泛采用的实验动物，目前国内外研究发现胚胎耳的发育绝大多数集中在E9以后。Ard认为，胚胎E9，E12，E14，E18为听功能发育的关键时期，故实验选取大鼠E9-E19期间标本。实验发现，大鼠内耳的发育开始于E8的外胚层增厚区，E9就可观察到听泡及其周围间充质的出现，E9.5听囊及听骨链开始发育，E12.5蜗管始基形成到E18.5耳蜗管发育完成。E16 Corti’s器发育形成，直到出生后内耳的结构和功能才发育完全。国外也有学者比较了人与几种哺乳动物的胚胎耳蜗各部的发育时间，发现人内耳的发育完成是W18，大鼠是E19。以上对大鼠胚胎耳蜗发育的研究为今后研究各种基因在内耳发育中的表达及作用提供了很好的形态学依据。
转化生长因子β是一种具有多功能活性的生长因子，参与了一些基本的生物过程，如细胞增殖、分化、凋亡、黏附、器官形成等^[3-14] 。转化生长因子β1是在人体中发现最广和研究最多的一种亚型，通过自分泌或旁分泌与靶细胞膜上的高亲和力受体结合而发挥其生物学作用^[19]。转化生长因子β1和转化生长因子β2对耳蜗的正常功能起重要作用^[20] 。Frenz等^[21]发现转化生长因子β1对内耳听软骨囊的形成有促进作用。Butts等^[22]证实胚胎期大剂量全反式维甲酸暴露的致畸作用与内源性转化生长因子β1减少有关，如果补充外源性转化生长因子β1对听软骨囊发育抑制有一定治疗作用。Paradies 等^[23]发现转化生长因子β1在小鼠中晚期胚胎的中胚层及外胚层的一些组织结构中皆有阳性表达，而且转化生长因子β1分布的时空形式与胚胎组织的形态发生具有一致性，认为转化生长因子β1参与小鼠胚胎组织的发育。赵玉林等^[24]发现在小鼠胚胎发育的第15-19天，转化生长因子β1在耳蜗的感觉上皮、支持细胞、血管纹、前庭膜、螺旋神经节神经元及椭圆囊斑和球囊斑的上皮均有阳性表达，认为其参与了小鼠内耳的发育过程并且主要是对内耳上皮的发育起促进分化作用。Kawamoto等^[25]用RT-PCR技术证实鼠耳蜗中存在转化生长因子β1和胶质细胞源性神经营养因子受体，且腺病毒介导的转化生长因子β1和胶质细胞源性神经营养因子表达可对氨基糖苷类耳毒性导致的毛细胞和听功能受损具有保护作用。Malgrange 等^[26]发现成年豚鼠Corti器培养时外毛细胞受损可用几种生长因子治疗，其中之一便是转化生长因子β1。Kim等^[27]利用免疫组化方法检测E13，E15，E17，E19 SD大鼠胚胎内耳转化生长因子βs的表达，结果E13时转化生长因子β1弱阳性表达，E15以后均为阴性，E15-E19转化生长因子β2表达均为阳性，且半规管壶腹、蜗管最强，转化生长因子β3仅在E15时表达阳性。本研究动态观察E9-E19大鼠胚胎耳蜗中转化生长因子β1的表达，结果发现，转化生长因子β1在此期间在不同部位均有不同强度的表达，胚胎大鼠内耳发育的中期，在螺旋神经节的神经细胞、耳蜗管的上皮细胞、支持细胞均有阳性表达，尤其是在听囊上皮细胞表达更明显；E14.5-E16.5耳蜗管上皮细胞、螺旋韧带、血管纹、基底膜存在转化生长因子β1阳性表达，而此时正是胚胎内耳上皮发生分化的时期。E18-E19耳蜗底周上皮表达已减弱，而顶回上皮及螺旋神经节的神经细胞仍有较强的表达，这与耳蜗上皮从底周向顶周分化发育的特点相一致。本文结果与文献不太一致的原因可能与动物品系、取材部位、检测技术等不同有关。
综上所述，转化生长因子β1在大鼠胚胎内耳分化期有阳性表达，且表达具有明显的阶段性，说明其可能参与内耳的发育过程，且主要对内耳上皮的发育起促进分化作用，但要确定转化生长因子β1在耳蜗发育过程中的具体作用及机制，尚需进一步研究。