细胞骨架细丝蛋白A在胚胎干细胞向早期神经细胞分化中的作用及其作用机制

Gene mutation is one of the major causes that lead to congenital genetic diseases. Mutations of cytoskeletal protein filamin A (FLNA) cause a number of organ anomalies including brain, heart and skeleton。One of congenital diseases, PVNH (Periventricular Nodular Hetrotopia) that clinically manifests brain anomaly is caused by abnormal neural development elicited by the mutations of FLNA actin-binding domain. However, the mechanism that FLNA mutations impair neural development is not fully understood. This project is aimed to investigate the role of FLNA in the differentiation of embryo stem cell to early neural cell and its acting mechanism using embryo stem as a model. In the project we will address the following questions: 1) Whether FLNA modulates the differentiation of mouse embryo stem cell to early neural cell? 2) How does FLNA modulate the differentiation of embryo stem cell to early neural cell? 3) Whether FLNA modulates the differentiation of human embryo stem cell to early neural cell? Addressing these questions will broaden our understanding to congenital disease PVNH caused by FLNA mutations and establish the basis for clinical intervention and prevention for the disease.

基因突变是先天性遗传疾病发生的主要原因之一。细胞骨架细丝蛋白A (Filamin A, FLNA)基因突变导致多种器官异常包括脑，心脏和骨骼发育畸形。其中，临床上表现为脑畸形的先天性疾病PVNH (Periventricular Nodular Hetrotopia)是由FLNA肌动蛋白结合域突变引起的神经发育异常所产生的。但是，FLNA突变阻碍神经发育的机制仍不完全清楚。本项目以胚胎干细胞为模型研究FLNA在胚胎干细胞向早期神经细胞发育中的作用及其作用机制。本项目将主要解答以下问题：1）FLNA是否调节小鼠胚胎干细胞向早期神经细胞分化？2）FLNA如何调节胚胎干细胞向早期神经细胞分化的？3）FLNA是否调节人胚胎干细胞向早期神经细胞分化？这些问题的解答可拓宽我们对FLNA突变导致的先天性疾病PVNH的了解，将为临床上干预和预防PVNH奠定理论基础.

细丝蛋白 A(FLNA) 是一种肌动蛋白结合蛋白，由X染色体上的基因编码，分子量为280KD, 参与细胞内多种生理生化活动，包括基因转录、信号转导、细胞增殖等。以前的研究表明FLNA基因突变可导致脑发育畸形，但FLNA是否对胚胎早期的神经分化和发育起作用还不清楚。本研究中，我们用小鼠胚胎干细胞A2lox和129，分别构建了FLNA knock-down稳定表达细胞系A2lox-FLNA shRNA、A2lox- Ctrl shRNA、129-FLNA shRNA和129-Ctrl shRNA，并用拟胚体和retinoic acid法诱导其向神经细胞的分化。我们的结果表明：FLNA knock-down可抑制小鼠胚胎干细胞向神经干细胞的分化，并进而抑制神经干细胞向神经元的分化及成熟；而FLNA knock-down可促进胚胎干细胞向星型胶质细胞的分化。利用所获得的稳定细胞系A2lox-Ctrl shRNA和A2lox-FLNA shRNA完成了全转录组测序分析，GO聚类分析表明FLNA knock-down影响胚胎干细胞中神经分化相关基因的表达，分析这些基因涉及的信号通路，发现很多差异表达基因参与了胚胎发育过程中的背腹轴形成以及线粒体能量代谢过程，这可能是FLNA参与调控胚胎干细胞向神经细胞分化的内在机理。这项发现不仅拓展对细胞中 FLNA 功能的了解，也为 FLNA影响脑发育的机制提供全新的解释。