高糖环境导致胚胎神经管缺陷的机制研究

Both gestational diabetes and diabetic pregnancy can result in the elevated glucose concentration in the environment of embryonic development. It has been proved that maternal hyperglycemia can cause many abnormalities such as Neural tube defects (NTDs) in central nervous system. NTDs are referred to the malformation in early neural development due to the disturbed interaction between genetic and external environment. It is reported that the maldevelopment of neural crest derived from dorsal side of neural tube is also an important reason for NTDs. However, the mechanism of NTDs induced by hyperglycemia remains obscure..In this study, we will investigate the cellular and molecular mechanisms of the abnormal development of the neural tube and neural crest induced by hyperglycemia in the early embryo. First, combining the advantages of both diabetic mouse and chick embryo models, we will investigate the expression pattern and quantity of related important genes on neural tube and neural crest at mRNA and also protein levels by making use of in situ hybridization, immunofluorescence, qRT-PCR and Western blot. Moreover, we will detect the cell cycle, proliferation, apoptosis, autophagy, oxidative stress and migration during neurulation. Meanwhile, combining the transgenic mouse models, we are capable of exploring the molecular mechanisms, so that a hypothesis can be pointed out about the role of maternal hyperglycemia in affecting neural tube and neural crest development, which might be able to provide theoretical basis of the prevention and treatment for NTDs in early gestation.

近年来，妊娠糖尿病或糖尿病合并妊娠的发病率呈上升趋势。两者都会导致胚胎发育直接暴露在高糖环境中，从而导致多种胚胎发育畸形，主要表现在中枢神经系统会出现神经管缺陷（NTDs）。NTDs的产生除神经发育异常外，神经管背侧神经嵴发育异常也是NTDs的一个重要因素，而高糖环境影响神经和神经嵴发育的机制仍远未得以阐明。因此，我们将结合高糖鼠胚、鸡胚模型的优势，从高糖环境导致的NTDs表型入手，利用原位杂交、免疫荧光、qRT-PCR以及Western blot等技术分别对神经和神经嵴发育以及神经管闭合相关关键基因的表达进行分析。此外，我们将检测高糖环境中神经和神经嵴细胞的细胞周期、自噬、氧化应激以及细胞迁移等活动，进一步探索高糖导致NTDs的细胞生物学机制。同时，结合相应转基因鼠，提出高糖导致NTDs产生的细胞和分子生物学假说，为早期妊娠过程中胚胎NTDs的预防和治疗提供理论基础。

妊娠糖尿病增加胚胎神经管畸形的发生，但其致畸的具体机制尚未完全明确。利用链脲佐菌素建立妊娠糖尿病（PGDM）的小鼠动物模型，体外高糖培养SH-SY5Y和U87细胞和鸡胚实验，分析高糖和对照组胚胎形态学改变，通过免疫组化、RT-PCR及western blot等方法检测神经元及神经胶质细胞的分化，检测TUNEL、PCNA及caspase家族分析细胞增殖和凋亡，通过SOD酶活性、Nrf2及其下游抗氧化因子的检测探讨高糖对细胞氧化应激和抗氧化反应的影响， 利用双色免疫荧光共定位神经元Nrf2的表达后，Nrf2-sh质粒转染SH-SY5Y神经元细胞降调Nrf2的表达，探索高糖环境下细胞活力的改变。发现高糖等应激环境通过氧化应激导致神经管闭合缺陷，诱导细胞自噬影响胚胎早期发育，对神经管发育过程中背腹侧基因的表达模式产生影响，对神经发生过程中影响EMT黏附分子和mTor信号。这些研究一定程度上揭示了高糖环境对神经管导致神经管缺陷和神经嵴细胞发育异常，与其改变细胞周期、氧化应激、自噬和细胞迁移等活动密切相关。相关研究成果以（共）通讯作者发表SCI论文18篇；项目资助指导毕业博士研究生1名，硕士研究生5名；研究成果在第七届亚太国际解剖会议（2016，新加坡），国际胚胎学会议（2017，美国）等作国际交流。