BMP2、BMP7/ Smad1通路是小鼠下颌骨继发性软骨软骨内成骨的“制动器”

Mandibular secondary cartilages include the condylar, coronoid, angular and symphyseal cartilage, which strongly influence the late development of the mandible. The purpose of this study is to investigate the temporospatial expression pattern and functional significance of BMP2 and BMP7 in seondary hypertrophic cell zone during the late development of mouse mandible. Our previous immunohistochemical examinations of BMP2, BMP7 and Smad1 were conducted in the mandibular secondary cartilages of the 15 neonatal C57BL/6N mice. In angular cartilage and symphyseal cartilage, surprisingly, BMP2, BMP7 and Smad1 were expressed in the entire hypertrophic cell zone of angular cartilage, which is distinct from that in the condylar cartilage. Consequently, it is suggested that the canonical BMP signaling pathway played a different role during the development of newborn mouse mandibular secondary hypertrophic cell zone. Thus, we want to thoroughly examine the temporospatial expression pattern of BMP2, BMP7 and Smad1 in mouse mandibular secondary hypertrophic cell zone with age by immunohistochemistry and analyzed the effects of BMP2,BMP7 and BMP antagonists noggin on the growth and development of mouse mandibular condylar cartilage and angular cartilage using serum-free organ culture and on the expression of genes related to the chondrocyte hypertrophic differentiation and maturation.

研究表明四个继发性软骨（髁突软骨、喙突软骨、角突软骨、正中联合处软骨）的软骨内成骨对下颌骨的发育起到至关重要的作用。肥大软骨细胞是骨生长的“引擎”，能够“发动”软骨内成骨。BMP2和BMP7在胚胎干细胞及间充质干细胞的成软骨和成骨诱导分化方面功能强大。我们的前期实验发现BMP2、BMP7和Smad1蛋白在新生小鼠髁突软骨肥大区未见表达，而在角突软骨和正中联合处软骨肥大区高表达，因此我们推测BMP2、BMP7/Smad1信号可能是调控继发性软骨细胞肥大分化速率的主因。本实验拟通过免疫组化探索BMP2、BMP7、Smad1在小鼠继发性软骨的时空表达特征，观察BMP2、BMP7、BMP细胞外拮抗剂Noggin对体外培养的髁突软骨和角突软骨肥大区的影响，试图明确其调控继发性软骨肥大分化的机制，为今后BMP2和BMP7应用于治疗继发性软骨，尤其是髁突软骨，发育异常这一设想提供实验理论依据。

研究表明继发性软骨的软骨内成骨对下颌骨发育起到至关重要的作用。肥大软骨细胞是骨生长的“引擎”，能够“发动”软骨内成骨。本课题完成免疫组化检测BMP2、BMP7和Smad1在小鼠继发性软骨中的时空表达：E16.5, BMP2和BMP7、Smad1均表达于正在分化的增殖细胞中，细胞快速分化。Day1，BMP2、BMP7和Smad1在髁突肥大软骨区未见表达，角突和正中联合的肥大软骨区可见表达；Day7，BMP2、BMP7和Smad1在髁突软骨肥大区出现表达，D21，三者在髁突软骨肥大区高表达，随后表达开始减弱。同时完成体外培养E16.5小鼠下颌骨。Day5，BMP2蛋白组髁突软骨变宽（近远中方向）, 且软骨下区（骨化）所占比例增大；BMP7蛋白组髁突肥大软骨区宽度增大，且软骨下区（骨化）略增大；BMP2和BMP7蛋白组髁突软骨宽度增加显著，骨化区域增多；Noggin蛋白组髁突软骨区明显变小，肥大软骨区也显著变小，成骨不连续。随后，通过Real-Time PCR检测BMP2、BMP7和Noggin 对体外培养髁突软骨的影响：BMP2蛋白组及BMP2和BMP7蛋白组中Col2a1和Col10a1、ALP和MMP13、Sox9和Runx2、Smad1和Smad4及PCNA均增高，具有统计学意义；BMP7蛋白组中Col2a1和Col10a1、Smad1和Smad4、Sox9和PCNA表达增高，具有统计学意义。Noggin蛋白组软骨细胞中Col2a1和Col10a1、ALP和MMP13、Sox9 和Runx2及PCNA 降低显著，具有统计学意义。明确BMP2可主要通过Smad1调控Runx2加速髁突肥大软骨细胞胶原Col10a1合成，BMP7可主要通过Smad1调控Sox9加速软骨特异性胶原Col2a1合成，两者可以协同调控继发性软骨肥大分化的速率。为今后BMP2和BMP7应用于治疗继发性软骨，尤其是髁突软骨，发育异常这一设想提供实验理论依据。