F-actin结合蛋白在维甲酸诱导的舌肌发育不良中的作用及机制研究

Proper tongue development is crucial to the maxillofacial system, and tongue abnormalities are correlated with maxillofacial malformations of human, until now, the pathogenesis and molecular details of abnormal development in tongue remain poorly understood. In our current research, we find that pregnant mice treated with retinoic acid (RA) produce embryos with tongue dysplasia; the analysis of DNA microarray showed that F-actin binding proteins Rai14, DMD and DTNA that are related to myogenesis and myogenic abnormality, are all dysregulated in RA induced tongue malformations, this indicated that Rai14, DMD and DTNA are involved in RA induced tongue dysplasia. On this basis, the project will firstly determine the spatial-temporal dynamic changes of DTNA, DMD and Rai14, and their correlation with proliferation and differentiation during tongue development using immunofluorescence staining. Furthermore, by the loss of function and gain of function studies using inhibitors, siRNA interference or recombinant proteins in primary cell culture of tongue myoblasts and tongue organ culture system, the changes in subcellular localization of protein, F-actin density and distribution, as well as ultrastructural analysis of tongue myoblasts will be explored. Then the interaction of RA, DMD，DTNA and Rai14, and how this network regulates tongue myogenesis will be well illustrated. This research provides a further realization to the pathogenesis and mechanisms of tongue abnormal development and tongue myogenesis, and provides a theoretical basis for the early diagnosis, prevention and clinical treatment of ralated diseases.

舌的正常发育对口颌系统的发育至关重要，舌发育异常与多种口颌畸形密切相关，但目前舌发育异常的病因和机制不清。本课题组前期研究发现维甲酸可导致舌肌发育不良；基因芯片结果显示，与肌肉发育及发育异常密切相关的细胞质骨架F-actin结合蛋白Rai14、DTNA及DMD在维甲酸诱导的舌畸形中表达异常，提示其与舌发育异常相关。本课题拟利用维甲酸诱导的小鼠舌畸形模型，采用免疫荧光染色等方法研究DMD，DTNA和Rai14时空表达及与增殖和分化的相关性；体外采用抑制剂、siRNA干扰或重组蛋白在原代培养细胞及体外培养的器官进行丧失性及获得性功能研究，通过检测蛋白亚细胞定位、细胞超微结构、F-actin动力学等揭示RA与DMD、DTNA、Rai14对舌肌发育的影响及相互作用的分子机制。该研究将有助于揭示舌发育及发育异常的病因及分子机制，为舌发育异常等口颌畸形的早期诊断、预防和治疗提供理论依据。

舌的正常发育对口颌系统的发育至关重要，舌发育异常与包括腭裂在内的多种颅颌面部畸形密切相关。舌肌属于骨骼肌，来源复杂，由于其成肌细胞的来源与躯干及四肢骨骼肌类似，因此舌肌发育的研究对躯干及四肢骨骼肌的研究有一定的借鉴作用，但调控舌发育的分子机制却知之甚少。本研究首先考察了维甲酸（RA）诱导腭裂及舌发育畸形的小鼠模型的表型以及相关因子的时空动态变化，发现过量维甲酸会导致胚胎舌体和颏舌肌萎缩及肌纤维坏死；在舌肌增殖特化的高峰期以及分化期，RA细胞核内受体RAR-alpha及RAR-beta表达变化，差异microRNA即miRNA-31-5p、DMD（dystrophin）以及Rai14均被异常调节；胎鼠出生前，DMD蛋白在腭裂组的舌体及颏舌肌里表达明显下降或表达缺失。其次利用体外细胞成肌分化模型进行丧失性及获得性功能研究，证实RA-miRNA-Dmd轴在成肌细胞的增殖发育过程中发挥重要作用，最终在RA处理后，通过抑制miRNA-31-5p的表达，上调Dmd表达，挽救了成肌细胞的增殖下降。本项目利用维甲酸诱导小鼠舌发育畸形模型，在国内外首次提出RA-RA receptor-miRNA-DMD的作用轴在腭裂伴发舌发育畸形中发挥作用。研究提示miRNA-31有可能作为疾病治疗效果的标记物；将来改善dystrophin的功能有可能成为治疗舌发育异常的切入点，促进腭裂患者语音功能的恢复；同时也提示人类杜氏肌营养不良症有可能与RA-RA receptor-microRNA-DMD的作用轴相关。本研究从miRNA及基因水平探究舌肌发育异常的分子调控机制，为筛查诊断以及未来治疗腭裂伴发舌肌发育不良的疾病提供了新思路，也通过寻找调控骨骼肌发育的关键性分子，为肢体骨骼肌发育疾病的预防和临床治疗提供科学依据。