TSC1通过GM130-Cdc42调控颅底软骨联合的发育及机制研究

Tsc1基因突变导致mTORC1信号持续上调，临床表现为多器官结节性硬化症（TSC）。我们通过建立TSC颅面部病变小鼠模型，发现在神经嵴来源的细胞中敲除Tsc1导致颅面畸形，其原因为颅底蝶骨间联合（ISS）过早融合。进一步发现，ISS早闭呈现统一的方向性，且软骨联合细胞中高尔基体蛋白GM130表达及分布改变。已知GM130调控下游蛋白Cdc42进而影响细胞迁移和极性。由此我们提出假说：Tsc1基因敲除导致颅底软骨联合过早闭合，其机制可能与GM130-Cdc42介导的软骨联合细胞极性和迁移方向改变有关。为验证假说，本研究拟通过形态学测量、细胞和分子生物学等方法，分层次研究Tsc1敲除对小鼠颅面部形态的改变、颅底软骨联合细胞发育的影响及细胞极性和迁移的分子调控机制等内容，旨在揭示Tsc1/mTOR在颅底生长发育中的作用，探究Tsc1与GM130的互作关系，为颅面畸形患者的正畸治疗提出新思路。

Tsc1 is the key negative regulator of mTORC1 signaling. Tsc1 mutation leads to hyperactivation of mTORC1, which manifesting as a multisystem syndrome called Tuberous Sclerosis Complex (TSC) in human. However, the mechanism and effective orthodontic therapy of craniofacial lesion of TSC syndrome is largely unknown. By generating a Tsc1 conditional knockout mouse model, we showed Tsc1 deletion in neural crest derived cells led to craniofacial deformity due to the premature fusion of inter-sphenoidal synchondrosis (ISS), which was in a specific pattern. Furthermore, the expression and distribution of GM130, a cis-Golgi matrix protein, altered in the resting zone chondrocytes in CKO mice. It was reported that GM130 regulated downstream small G-protein Cdc42 and therefore affect cell polarity and migration. Herein, we hypothesize that the premature fusion in Tsc1-deficient ISS may result from chondrocyte polarity and migration alteration mediated by GM130 and Cdc42. To further determine the mechanism, we designed following plans using our established knockout mouse model. Firstly, we will characterize the three-dimensional craniofacial deformity and ISS fusion pattern in CKO mice using micro-CT and morphological analysis. Secondly, the proliferation, differentiation and apoptosis of synchondrosis chondrocytes will be analyzed using cytobiological approaches. Lastly, the mechanism in that how Tsc1/mTORC1 signaling affect cell polarity and migration via GM130-Cdc42 will be investigated using molecular biological techniques involving in vivo and in vitro experiments. This study is aiming to promote a better understanding in the role of Tsc1/mTORC1 signaling in craniofacial development and give new insights in the clinical targeted-therapy for the patients with TSC-syndrome.