【摘要】 小鼠胚胎干细胞（mouse embryonic stem cells, mESCs）凭借其多向分化潜能和自我更新特性，已成为解析哺乳类胚胎发育机制的理想模型。叶酸作为一碳代谢的核心辅酶，其缺乏导致的神经管缺陷（neural tube defects, NTDs）已成为全球性公共卫生问题^[1],但其致病机制不明。叶酸是否会通过影响小鼠胚胎干细胞向神经谱系分化进而参与NTDs疾病的发生尚不明确。本研究通过构建叶酸缺乏mESCs向神经谱系分化模型，系统解析叶酸在神经谱系定向分化中的作用及其与神经管畸形发生的潜在关联。研究结果显示，在mESC向神经谱系分化过程中，叶酸缺乏mESCs诱导的拟胚体直径显著小于正常叶酸对照组（P<0.05）;通过实时荧光定量PCR（real-time quantitative PCR, RT-qPCR）及免疫荧光检测神经前体细胞标志物巢蛋白（nestin）的表达水平，结果显示，相较于正常叶酸对照组，叶酸缺乏组巢蛋白的mRNA水平和蛋白质水平均下降（P<0.05）。此外，免疫荧光染色也显示Ki67荧光水平在叶酸缺乏组明显下降（P<0.05）,提示叶酸缺乏诱导分化的神经前体细胞增殖能力受损。同时，在神经分化过程中的关键时间节点第4 d（day 4, D4）、第8 d（day 8, D8）分别对其进行转录物组测序。分别对比对照组和叶酸缺乏组在D4和D8的差异表达基因（differentially expressed gene, DEGs）进行GO功能富集，结果提示，叶酸缺乏导致中胚层分化偏倚，提示叶酸缺失可能通过破坏胚层命运决定导致神经分化异常。比较D4和D8的对照组和叶酸缺乏组DEGs, KEGG通路分析进一步揭示PI3K-AKT、MAPK及Wnt信号通路在叶酸缺乏组显著富集。Western印迹检测PI3K、AKT及其磷酸化蛋白质水平在叶酸缺乏组的D8显著升高，使用PI3K抑制剂可使叶酸缺乏组Nestin、Pax6、Otx2表达水平显著升高（P<0.05）,可挽救叶酸缺乏导致的外胚层分化下降现象。综上，本研究通过构建叶酸缺乏小鼠胚胎干细胞分化模型，揭示叶酸在神经谱系命运决定过程中的作用，阐明叶酸缺乏导致的谱系命运决定异常可能是NTDs发生的原因之一。本文的研究为阐释叶酸缺乏导致神经管畸形的致病机制提供新视角，也为相似发育缺陷的致病机制研究提供新的理论依据。更多还原

【Abstract】 Mouse embryonic stem cells(mESCs) have become an ideal model for analyzing the developmental machinery of mammalian embryos due to their multidirectional differentiation potential and self-renewal properties. Folate, a core coenzyme in one-carbon metabolism, has become a global public health problem due to its deficiency in neural tube defects(NTDs), but its role in the neural pedigree differentiation of mESCs is not clear. In this study, we systematically analyzed the regulatory mechanism of folate in neural lineage differentiation and its potential association with the development of neural tube deformities based on the neural differentiation model of ESCs. Research results showed that during the differentiation of mESCs into the neural lineage, folate deficiency led to a significantly smaller diameter of embryoid bodies compared with the normal folate control group(P < 0.05). The expression level of Nestin, a neural precursor cell marker, was detected by real-time quantitative PCR(RT-qPCR) and immunofluorescence. The results showed that both the transcriptional and protein expression levels of Nestin in the folate deficiency group were decreased compared with the normal folate control group(P < 0.05). In addition, immunofluorescence staining also showed that the Ki67 fluorescence level was significantly decreased in the folate deficiency group(P < 0.05), indicating that folate deficiency impairs the proliferation ability of neural precursor cells. Meanwhile, transcriptome sequencing was performed on the key time points of neural differentiation, day 4(D4) and day 8(D8). The GO functional enrichment analysis of differentially expressed genes(DEGs) between the control group and the folate deficiency group at D4 and D8 respectively revealed that folate deficiency caused a bias in mesoderm differentiation, suggesting that folate deficiency may lead to abnormal neural differentiation by disrupting the balance of germ layer fate determination. The KEGG pathway analysis of DEGs between the control group and the folate deficiency group at D4 and D8 further revealed that the PI3K-AKT, MAPK and Wnt signaling pathways were significantly enriched in the folate deficiency group. Western blotting showed that the protein levels of PI3K, AKT and their phosphorylated forms in the folate deficiency group were significantly increased at D8. The use of PI3K inhibitors could significantly increase the expression levels of Nestin, Pax6 and Otx2 in the folate deficiency group(P < 0.05), thus reversing the impairment of ectoderm differentiation caused by folate deficiency. In this study, we constructed a model of the dynamics of ESC differentiation due to folate deficiency to reveal the role of folate in the process of induced differentiation of neural progenitors, which may be one of the mechanisms by which folate deficiency leads to the development of NTDs. Our study provides new perspectives for elucidating the pathogenic mechanism of neural tube malformations caused by folate deficiency, as well as a new theoretical basis for the pathogenic mechanism of similar developmental defects.更多还原