肌醇营养物失衡经PI3K/AKT通路异常致神经管畸形的作用机制

Inositol deficiency in the periconceptional is closely related to the incidence of neural tube defects (NTDs), and the exact mechanism is unclear. Previous studies have shown that intracellular level of inositol in cancer cel lregulates the PI3K/Akt pathway activity, and the abnormal activated the PI3K/Akt pathway could affect the primary cilium formation during the embryonic development, while abmornalprimary cilium could lead to NTDs occurance. However, whether inositol nutrient imbalance effects on primary cilium and the neural tube formation through the PI3K/Akt pathway during the embryonic development has not been reported. Our recent study demonstrated that the low maternal inositol and its metabolic intermediates were associated with the occurrence of NTDs. The PI3K/Akt pathway activity is significant high among the NTDs embryonic brain tissues with low level of inositol. This study intends to inhibit the specific key enzymein inositol metabolism so as to establishthe animal models with inositol metabolic imbalances. Using the GC/MS, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), RNA - Seq and ChIP - qPCR methods, we plan to investigate the relationship between metabolic imbalance of inositol and PI3K/Akt pathway including downstream gene expression,in the inositol deficiency population and the established animal model. In order to validate the above results, the primary cultured mouse embryonic fibroblasts and neural stem cells will be used as study subject, to intend to get the abnormal gene expression profiling downstream of PI3K/Akt pathway under the inositol metabolic imbalances condition. The expected results will provide the theory evidence on clinical prevention of NTDs from the view of PI3K/Akt pathway.

围孕期肌醇缺乏与神经管畸形（neural tube defects，NTDs）发生密切相关，其确切机制尚不明确。研究发现肿瘤细胞内肌醇可调控PI3K/Akt通路活性，而该通路异常激活可影响胚胎初级纤毛形成；初级纤毛异常可致NTDs发生。肌醇代谢失衡是否通过该途径影响纤毛及神经管形成尚未见报道。前期研究发现NTDs孕妇血清肌醇及其代谢产物水平明显降低，低肌醇NTDs胚胎脑组织PI3K/Akt通路活性增高。本研究拟通过特异性抑制肌醇代谢靶点酶构建肌醇代谢失衡动物模型，用GC/MS、扫描电镜、透射电镜、RNA-Seq及ChIP-qPCR等方法，在低肌醇人群及动物模型研究肌醇代谢失衡与PI3K/Akt通路及下游基因表达关系，用原代小鼠胚胎成纤维细胞及神经干细胞验证，筛出PI3K/Akt通路异常激活的神经管闭合相关基因转录组，预期结果可为疾病预防及诊断从PI3K/Akt通路调控机制角度提供实验据。

围孕期肌醇缺乏与神经管畸形（neural tube defects，NTDs）发生密切相关，其确切机制尚不明确。研究发现肿瘤细胞内肌醇可调控PI3K/Akt通路活性，而该通路异常激活可影响胚胎初级纤毛形成；初级纤毛异常可致NTDs发生。肌醇代谢失衡是否通过此途径影响纤毛及神经管形成尚未见报道。课题组前期研究发现NTD孕妇血清肌醇及其代谢产物水平明显降低，低肌醇NTD胚胎脑组织PI3K/Akt通路活性增高。在此基础上，本研究选用肌醇代谢通路中关键酶肌醇-1-磷酸酶（IMPase）抑制剂为靶点，建立了小鼠肌醇代谢障碍NTD模型。利用已建立的GC/MS技术、扫描电镜、透射电镜、RNA-Seq、免疫组化、RT-PCR、蛋白质芯片技术及生物信息学等方法，发现叶酸水平正常的人类NTD样本及小鼠模型肌醇代谢失衡与PI3K/Akt通路异常激活；并用蛋白磷酸化芯片找到了人类及小鼠共同的Akt、mTOR、PTEN及p70S6K等14个磷酸化异常的关键蛋白质；肌醇失衡下小鼠胚胎神经组织细胞异常增殖；转录本（RNA）水平分析发现肌醇代谢失衡下NTD PI3K/Akt通路及初级纤毛形成关键基因（INPP5E、PTEN、PI3K及Akt1等）表达异常；细胞模型进一步验证肌醇代谢失衡下PI3K/Akt信号通路活性异常增高，纤毛数量明显减少，长度变短，纤毛相关蛋白INPP5E、α-tubulin等表达水平明显下降（P<0.05），提示肌醇失衡影响细胞初级纤毛形态及纤毛相关蛋白的表达。结果表明肌醇代谢失衡可能通过PI3K/Akt通路异常激活引起其下游关键基因的表达异常，影响胚胎神经发育过程中初级纤毛的形成导致NTD，为该病的早期诊断及防控，尤其是叶酸不敏感的NTD的早期诊断及防控，从营养素肌醇补充及PI3K/Akt通路调控机制角度提供实验依据，同时为NTDs发病分子机制的进一步阐明及合理化预防提供理论依据。