Rac1调控细胞迁移/分化在先天性小耳畸形发生中的作用和机制研究

Microtia is a congenital deformity where the external ear is underdeveloped. Cell migration plays an important role in the morphogenesis of tissues and organs. In our previous study,we found that the migration ability of chondrocytes from congenital microtia was significantly decreased compared with that of healthy auricular chondrocytes, which was characterized by a decrease in migration displacement and a random direction. Furthermore, the Rac1 gene, which is required for cell polarization and directional migration, has been detected decreased expression in the microtia chondrocytes using the Cell Motility PCR Array. Previous studies have confirmed the important role of Rac1 in cell migration and chondrocyte differentiation by regulating cytoskeleton reorganization. We thus establish Rac1 as a novel regulator of microtia development and elucidate the roles of its downstream signaling pathways at cellular and molecular levels. In the present study, we will firstly verify the reduced migration capacity and decreased Rac1 expression in microtia chondrocytes, and then establish the H9 ESC–derived NCC – MSC - chondrocyte differentiation model to examine the dynamic expression of Rac1 and elucidate its role in cell migration, proliferation, apoptosis and differentiation in different cell stage. Furthermore, we will confirm the role of Rac1 in external ear development using the mouse model of conditional deletion of Rac1 in NCC-derived MSC. Finally, the molecular mechanism will be clarified, by which Rac1 modulats cytoskeletal reorganizaiton and affects cell migration and differentiation. The results of the study may provide new insights into understanding the etiology of non-syndromic congenital microtia and potential new targets for clinical prevention and treatment.

细胞迁移对组织、器官的形态发生具有重要作用。我们前期研究发现小耳畸形软骨细胞迁移能力较正常耳软骨细胞显著降低，表现为细胞迁移位移减少和迁移方向紊乱；通过细胞能动性PCR芯片检测到与迁移极性定位密切相关的Rac1基因在残耳软骨中表达降低。Rac1是调节细胞骨架重组的关键基因，已证实在细胞迁移和软骨分化中均具有重要作用。本项目拟从细胞迁移角度出发，以Rac1为切入点，探讨通过调节细胞骨架重组调控细胞迁移和分化，影响外耳形态发生的作用。研究中，首先验证残耳软骨细胞的迁移能力和Rac1表达降低；然后应用人ESC来源的NCC-MSC-软骨细胞分化模型，明确Rac1影响外耳发育的细胞学机制；应用MPZ-Cre；Rac1fl/fl条件性基因敲除小鼠模型验证Rac1对外耳发育的作用；并从细胞骨架调控通路明确Rac1发挥作用的分子机制，为先天性小耳畸形的病因研究提供新思路，为临床防治寻找新靶点。

先天性小耳畸形（congenital microtia）是我国第二大颅面部先天性疾病，造成的耳廓畸形和听力障碍严重影响儿童的身心健康。明确小耳畸形的发病机制对于疾病的防治具有重要意义。细胞迁移对组织、器官的形态发生具有重要作用。本项目从细胞迁移角度出发，以Rac1为切入点，探讨其调控细胞迁移，影响外耳形态发生的作用。研究基本按计划执行，完成了 （1）先天性小耳畸形较正常耳软骨细胞的迁移能力和Rac1表达检测。结果显示，与正常耳廓软骨相比，小耳畸形软骨组织表现出明显的细胞排列不规则和细胞外纤维组织紊乱。迁移实验显示小耳软骨细胞迁移能力显著低于正常耳软骨细胞。高内涵成像分析和OrisTM细胞迁移实验评估细胞累计运动距离、迁移位移和平均运动速度，以及迁移轨迹直线率和旋转角度，证实了小耳畸形软骨细胞迁移方向持久性降低。细胞运动PCR芯片、Real-time PCR、Western blot和免疫荧光结果均证实Rac1、ENAH、VASP、MMP14和PXN在小耳软骨细胞中稳定低表达和定位异常。（2）Rac1影响细胞定向迁移的机制研究。不同构象的Rac1（总Rac1、Rac1-P、Rac1-GTP）在小耳软骨细胞中的表达异常，表现为总Rac1弥散性分布在小耳软骨细胞的胞浆中，Rac1-P则大量出现在细胞核中，而Rac1-GTP与细胞骨架的重构不同步。Rac1活化分子TIAM1、ARHGEF12、ABR和ARHGAP19等多个Rho GEF和Rho GAP家族成员在小耳软骨细胞中表达降低，表明小耳软骨细胞中的Rac1上游活化机制存在缺陷。过表达Rac1-Q61L和Rac1特异性活化因子Tiam1均能部分挽救小耳软骨细胞的定向迁移能力。证实Tiam1低表达导致活性Rac1（Rac1-GTP）降低是其影响小耳软骨细胞定向迁移持久性的机制；（3）在小耳畸形鼠模型中验证Rac1-Tiam1通路异常。Bmp5se/J 和Prkralear-3J/GrsrJ的纯合突变个体均表现出耳廓软骨组织的紊乱，Tiam1的表达缺陷，以及活性Rac1的表达不足。通过项目研究，证实细胞迁移是影响外耳发育的细胞学基础，明确了Rac1对外耳发育的影响及调控细胞迁移的机制，为其临床防治和分子筛查提供理论基础和潜在靶点，为提高组织工程软骨种子细胞质量提供线索。