视黄酸对ALX3的基因调控作用和分子机制研究

Alx3 encodes a nuclear protein with a homeobox DNA-binding domain that functions as a transcriptional regulator involved in cell-type differentiation and developmentuse. Abnormal in the expression and function of ALX3 gene can cause severe craniofacial and skeletal malformations. However,the exact function and the mechanism of ALX3 involved in the human embryogenesis remains to be elucidated. Human embryonic stem cell(ESCs) is a type of cell derived from inner cell mass of human embryo at blastocyst stage. Human ESC is pluripotent and has ability to self-renew indefinitely. Due to its unique properties, human ESC may be used as a new and effective model to study the mechanism of early human embryognensis in vitro. In the pilot study, for the first time, we found that the expression of ALX3 is strongly associated with the concentration of retinoic acid (RA) in a dose-dependent manner. RA is an active metabolite of Vitamin A (retinol). It plays a crucial role in the pattern formation and may be one of the morphogens that control embryonic development and human morphogenesis. Therefore, in this study, we use human ESC and its derivatives as study models to analyze the function and mechanism of RA in the regulation of ALX3 expression. We plan to use RT-PCR and western blot analysis to identifiy types of RA receptors and related signaling molecules present in these cell models after RA treatment followed by immunoprecipitation to purify those proteins. Next, the electrophoretic mobility shift assay (EMSA) will be used to identify the possible binding sites of the purified signal molecules in the ALX3 promoter region. This study may help us understand the function and the mechanism of RA in the regulation of ALX3 expression during the early human embryogenesis and may provide a theoretical basis for the prevention and treatment of ALX3-related diseases in future.

ALX3是一个胚胎发育相关基因，其表达水平及功能的异常往往会造成严重的颅面和骨骼发育畸形。目前，ALX3在人胚胎发育过程中的具体作用和相关分子机制尚不明确。人胚胎干细胞（hESC）来源于处于囊胚期的人胚胎内细胞团，是体外研究早期胚胎发育机制的一个理想模型。本课题组在前期研究中首次发现，hESC及其分化细胞中ALX3的表达水平与视黄酸（RA）紧密相关。因此，本项目拟采用hESC及其分化细胞作为模型，运用PCR和蛋白免疫印迹等方法，检测RA处理后细胞中表达的RA受体及相关信号分子的种类并利用免疫共沉淀等方法提纯相关蛋白，采用凝胶迁移等技术找出这些蛋白在ALX3启动子区域上可能存在的结合位点，再通过基因敲除和过表达等方法分析该信号分子在相关信号通路上发挥的充要作用，探讨RA在ESC及其分化模型中调控ALX3的信号通路和分子机制。本项研究可帮助我们了解RA在胚胎发育早期对ALX3的调控作用和机制。

视黄酸（RA）与胚胎的正常生长发育密切相关，过量RA摄入会导致唇腭裂等先天发育畸形。研究发现RA在胚胎发育早期主要作用在神经嵴细胞（NCC）上。由于缺乏合适的人来源研究模型，RA调控人胚胎发育的作用机制尚不明确。人胚胎干细胞（ESC）来源于处于囊胚期的人胚胎内细胞团，是体外研究早期胚胎发育机制的一个理想模型。本课题组在前期研究中首次发现，人ESC及其分化细胞中ALX3的表达水平与RA紧密相关。ALX3是一个胚胎发育相关基因，其表达水平及功能的异常往往会造成严重的颅面和骨骼发育畸形。目前，ALX3在人胚胎发育过程中的具体作用和相关分子机制尚不明确。因此，本项目采用人ESC及其分化的NCC为研究模型，探讨了RA对人ESC及其来源NCC的生物学作用及其对ALX3基因的调控作用和分子机制。研究发现RA可诱导人ESC发生分化干性降低，并诱导拟胚体发生过度分化，破坏EB分化早期的成骨和成脂分化平衡。此外，RA还可促进ESC来源NCC发生凋亡并通过抑制EMT的发生降低ESC来源NCC的迁移能力。定量real-time PCR检测结果证实RA可显著上调不同分化程度的人ESC来源细胞中ALX3基因的转录水平。机制研究结果显示RA对人ESC中ALX3转录水平的上调是由PAX3和HOXA5介导的。本项研究可帮助我们了解RA在胚胎发育早期对ALX3的调控作用和机制，为了解人胚胎发育早期的相关机制提供思路，为将来预防、诊断和治疗与RA和ALX3相关的颅颌面发育疾病提供参考和解决办法。