基于自噬途径的亲代高脂饮食对小鼠子代神经干细胞增殖分化作用机制的研究

The incidence of obesity and overweight has reached epidemic levels in the worldwide. Even more alarming is the increasing prevalence of metabolic diseases in younger children and adolescents. Infants born to obese, overweight have increased adiposity and are at increased risk of later metabolic disease. Hyperlipidemia and inflammation may contribute to the childhood obesity epidemic through fetal metabolic programming, the mechanisms of which are not well understood. Studies in animal models suggest that maternal obesity and high-fat diet consumption lead to impaired leptin signaling and adaptive regulation of key genes in neuronal pathways associated with appetite in hypothalamus. This suggests that maternal high-fat diet has profound effects on fetal brain development, and may impact neural stem cell, which warrant further investigation. Our preliminary data showed that expression of MTMR13 was much lower in high-fat-diet offsprings with hypermethylated promoter. MTMR13 is PI 3-phosphatase with specificity for PtdIns3P and PtdIns(3,5)-P2. PtdIns3P serves as a scaffold for the assembly of the autophagic apparatus required for limiting membrane formation, autophagosome trafficking in microtubules, lysosomal fusion, and even in cargorecognition. Autophagy is a lysosome-dependent degradation pathway that allows cells to recycle damaged or superfluous cytoplasmic content, such as proteins, organelles, and lipids. Deficiencies in this pathway were associated to several pathological conditions, such as neurodegenerative and cardiac diseases, cancer, and aging. Recent studies also show autophagy plays a critical role in stem cell maintenance and in a variety of cell differentiation processes. Therefore, this study will focus on the effects of maternal high fat diet on proliferation and differentiation of neural stem cell in CD1 mice. The mechanism of the central hypothesis will be explored including the involvement of MTMR13, PtdIns3P and macroautophagy by MTMR13 overexpression/knock-down, assessment PtdIns3P with GST-FYVE, neural stem cells stably expressing GFP-LC3 and live cell workstation. This study will define the molecular mechanism responsible for altered differentiation of neural stem cell leading to childhood obesity and metabolic symdrome. And this will provide a basis for setting dietary guidelines for pregnant women that may protect offspring from enhanced susceptibility to metabolic diseases.

儿童肥胖、糖尿病是影响儿童健康的严重卫生问题，研究证实亲代高脂饮食是主要原因。亲代高脂饮食促进胎儿海马区神经干细胞增殖、刺激胚胎促食欲神经元的增殖并导致瘦素信号通路的缺陷是子代肥胖的重要机制。前期研究发现高脂饮食孕鼠子代的自噬抑制基因MTMR13启动子甲基化明显提高、表达显著降低。自噬参与调控神经细胞脂类代谢以及干细胞分化。据此假设：亲代高脂饮食通过DNA甲基化机制抑制子代MTMR13表达、自噬增强，进而影响神经干细胞增殖和分化，最终干扰神经系统发育并影响胎儿发育。本研究拟检测高脂饮食对仔鼠NSC增殖、分化和自噬的影响，及机制探索：MTMR13过表达/抑制、自噬诱导/抑制对NSC的作用，利用稳定表达GFP-LC3细胞株，结合活细胞成像、激光共聚焦检测高脂血清、MTMR13过表达和敲减时的LC3、PtdIns3P及分化情况。将利于科学指导孕龄女饮食，有效预防儿童肥胖，为治疗提供新思路。

本研究旨在探索高脂饮食对子代神经干细胞的增殖、分化和自噬的影响。经过一年的努力，取得了实质性的研究进展，三篇SCI论文已被接收，影响因子总分值（IF）达到11。主要研究结果总结如下：1、成功建立了母体高脂饮食小鼠模型，交配前高脂组雌鼠体重增长、内脏脂肪堆积以及血清胆固醇水平都显著高于对照组；2、高脂饮食组子代神经干细胞的增殖（细胞计数、免疫细胞化学法）未见显著不同于对照，而其Notch信号通路关键基因Msi1、Msi2、Notch1的表达显著显著增加；3、运用MeDIP-chip技术检测高脂饮食对子代基因组甲基化的影响，结果显示：HFD组甲基化整体水平有所降低，利用QDMR软件筛选到315个HFD组和对照组间差异甲基化的区域（ROI），其中228个ROI在HFD组甲基化降低，96个升高。差异甲基化ROI在基因上游的比例显著减少，而在基因间区的比例显著增加。我们通过DAVID在线软件对这些基因进行了GO功能注释分析，结果显示这些基因显著富集在多种生物学功能。其中经过Benjamini校正的生物学功能有六个GO term：sequence-specific DNA binding、transcription factor activity、transcription regulator activity、DNA binding、regulation of RNA metabolic process、regulation of cell proliferation；4、高脂饮食对子代神经干细胞分化和自噬未见显著影响。