基于神经干细胞增殖分化母体叶酸缺乏对大鼠子代神经发育作用机制的研究

Folic acid supplement during pregnancy could decrease the prevalent of neural tube defects, improve neurodevelopment of offspring, and folic acid deficient during pregnancy could hinder neurodevelopment of offspring, however, the mechanism of how folic acid effect on neurodevelopment of offspring is still not clearly. In this study, animal reproductive study will be used to detect the effect of folic acid on neurodevelopment of offspring. Female SD rat will assigned in equal numbers to two groups for 90 days: (a) folate-deficient diet, (b) control diet (normal folic acid content). Mating after grouping 60 days, then pups will be getting after delivery. And then pups were randomly assigned to 2 parts: one part pups were killed to get brain tissue, one part pups used to culture neural stem cells (NSCs) in vitro. The newly born NSCs, and the proportion of neuron and neurogliocyte will be detected by immunohistochemical in vivo. The proliferation and differentiation of NSCs will also detect used culture NSCs in vitro. The microRNA chip will be detected at offspring brain tissues in vivo and culture NSCs in vitro. The study will elucidate the underlying mechanism of the effects of folic acid deficiency on neurodevelopment by the proliferation and differentiation of NSCs, and demonstrate the relationship of folate deficient, NSCs and neurodevelopment. The molecular mechanisms of the effects of folic acid on neurodevelopment by microRNA will be explored.

孕期叶酸补充可以有效减少神经管畸形的发病率，改善子代神经发育状况，叶酸缺乏会影响子代大脑神经系统发育，但是其作用机制仍不十分清楚。本研究采用大鼠生殖实验，将雌性大鼠随机分为叶酸缺乏组和叶酸正常组，分别饲喂叶酸缺乏饲料或叶酸正常饲料，检测子代鼠脑组织原位病理切片神经干细胞、神经元及神经胶质细胞的比例；体外培养子代鼠脑皮层神经干细胞检测细胞增殖分化情况；采用microRNA芯片结合表达谱芯片分别检测新生子代鼠大脑组织和体外培养子代鼠大脑皮层神经干细胞中microRNA的表达水平。探讨叶酸缺乏和蛋氨酸循环代谢、神经干细胞增殖分化和神经发育之间的关系，明确基于神经干细胞增殖分化叶酸缺乏在神经发育中的作用机制，确定叶酸缺乏-神经干细胞-神经发育三者之间关系，为探讨叶酸缺乏在神经发育过程中分子机制及叶酸防治神经缺陷相关疾病提供科学依据。

神经干细胞（NSCs）的增殖、分化及凋亡过程在胚胎神经发育中起到了至关重要的作用，叶酸是否通过调节NSCs增殖、分化及凋亡过程对子代神经发育产生作用及其机制尚不清楚。本研究采用大鼠生殖实验，模拟人孕期叶酸营养状态，将雌性SD大鼠分为叶酸缺乏组和叶酸正常组，分别饲喂叶酸缺乏饲料（含0.1mg叶酸/kg饲料）和叶酸正常饲料（含2.1mg叶酸/kg饲料）60天。至雌性大鼠约14周龄，将雌鼠与雄鼠按4：1合笼交配。孕鼠怀孕生产期间继续饲喂本组对应饲料，待受孕雌鼠生产后，获得子代鼠。收集母鼠不同时期血液样本，子代鼠脑组织，观察评估子代鼠神经发育状况。研究结果提示：母体孕期叶酸缺乏抑制子代大鼠神经发育，其可能的机制是①孕期叶酸缺乏降低子代脑组织DNA甲基化水平并使子代脑组织神经细胞超微结构发生不利改变；②孕期叶酸缺乏上调microRNA-34a，启动细胞凋亡通路引起子代神经细胞凋亡。体外实验进一步证实叶酸调控notch信号通路提高体外培养的NSCs增殖活力；叶酸保护氧化应激导致的端粒磨损减少星形胶质细胞凋亡。研究结果可为探讨孕期叶酸缺乏引起子代神经发育延迟的机制提供科学依据，具有理论和实用意义。