高糖对早期鹌鹑胚胎神经系统发育的影响

发布时间：2018-01-16 08:11

本文关键词：高糖对早期鹌鹑胚胎神经系统发育的影响　出处：《暨南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：孕期妊娠糖尿病的定义是葡萄糖耐受不良，表现为孕妇在怀孕期间血糖水平升高的现象。然而这种情况下对母体内胎儿的发育有不利影响，但是它所涉及的机制仍不完全清楚。在这项研究中，我们以鹌鹑胚胎为模型，研究了高葡萄糖环境下对鹌鹑胚胎发育的影响，特别是对胚胎神经系统发育的影响。方法和结果：我们通过HE染色证实了高糖环境下可以损害中枢神经系统，造成神经管出现不闭合的缺陷。此外，我们通过NF免疫荧光和镀银染色发现高糖处理后胚胎的背根神经节以及肢芽处的神经元细胞发育都受到了明显抑制的现象。出现了背根神经节尺寸和数目的减少。我们了解到由于背根神经节来源于一部分神经嵴细胞的迁移和分化，所以我们探究高糖是否对从神经管背侧迁移出来的神经嵴细胞的迁移量有影响。我们用HNK-1免疫荧光的方法在体内和体外神经嵴细胞的迁移实验证实高糖对神经嵴细胞的迁移作用。结果显示高糖的确对神经嵴细胞的迁移有明显的抑制作用。包括迁移量减少以及HNK-1阳性表达变弱。最后，我们经过添加高糖处理的培养基通过体外培养神经元细胞的实验探究高糖对神经元细胞的损害作用。研究结果表明与正常培养基中的神经元细胞相比较，高糖培养基中神经元的数目会变少，神经元轴突的长度变短以及培养的神经元细胞的胞体折光度变弱。随后我们检测了神经元细胞的活性氧自由基(ROS)的产生量，结果显示ROS会随着高糖的浓度增加而增加。于是我们假设，过量的活性氧自由基是神经元的发育和分化异常的主要因素，如果除去神经元细胞产生的过量的ROS，神经元的生长状况会不会有所改善。因此我们往高糖培养基中加入一定量的维生素C(抗氧化剂)，再次测定神经元的数目、轴突的长度以及神经元细胞的活性氧自由基(ROS)。我们的假说得到了证实，结果表明通过添加维生素C可以一定程度的改善高葡萄糖对神经元发育的损坏性影响。结论：我们以上的研究结果表明高糖对神经管，，背根神经节和神经在肢芽处的发育都有一定程度的损害作用。然而这些损害作用都是由于高糖过剩引起神经元细胞内的ROS的增加，继而影响神经嵴细胞的迁移以及分化而导致的。然而我们在培养的神经元细胞的高糖培养基中添加了抗氧化剂维生素C后，维生素C可以减少ROS的产生，神经元细胞的生长状况也得到了一定的改善。它意味着维生素C不是直接保护细胞发挥其作用而是通过抑制ROS的产生而对神经元起到保护作用。
[Abstract]:Objective: gestational diabetes mellitus is defined as impaired glucose tolerance, which is characterized by increased blood glucose levels during pregnancy. However, this condition has adverse effects on maternal fetal development. However, the mechanism involved is still unclear. In this study, we used quail embryos as a model to study the effects of high glucose on the development of quail embryos. In particular, the effects on the development of the embryonic nervous system. Methods and results: we proved by HE staining that high glucose environment can damage the central nervous system, resulting in the defect of the neural tube is not closed. Through NF immunofluorescence and silver staining, we found that the development of neurons in dorsal root ganglion and limb bud of embryos treated with high glucose was obviously inhibited. The size and number of dorsal root ganglion decreased. We understand that the dorsal root ganglion is derived from the migration and differentiation of some neural crest cells. So we explored whether high glucose had an effect on the migration of neural crest cells from the dorsal side of the neural tube. We used HNK-1 immunofluorescence assay in vivo and in vitro to confirm the migration of neural crest cells. The results showed that high glucose did inhibit the migration of neural crest cells, including the decrease of migration amount and the weakening of HNK-1 positive expression. The effects of high glucose on neuronal cells were investigated by the experiment of cultured neurons in vitro. The results showed that the effects of high glucose on neuronal cells were compared with those in normal culture medium. The number of neurons in high glucose medium will decrease. The length of neuronal axons became shorter and the somatic diopter of cultured neuron cells weakened. Then we measured the production of reactive oxygen species (Ros) of neuron cells. The results showed that ROS increased with the increase of high glucose concentration, so we hypothesized that excessive Ros was the main factor of abnormal development and differentiation of neurons. If the excess ROSs produced by neuron cells were removed, the growth of neurons would be improved, so we added a certain amount of vitamin C (antioxidants) to high glucose medium. The number of neurons, the length of axons and the reactive oxygen species (Ros) of neuronal cells were determined again. Our hypothesis has been confirmed. The results showed that vitamin C could improve the damage effect of high glucose on neuron development to some extent. Conclusion: the results of our above studies show that high glucose on the neural tube. The development of dorsal root ganglion (DRG) and nerve in limb bud are damaged to some extent, however, these damages are due to the increase of ROS in neuronal cells caused by high glucose excess. However, vitamin C can reduce the production of ROS by adding vitamin C to the high sugar medium of cultured neuron cells, which affects the migration and differentiation of neural crest cells. The growth of neuronal cells has also been improved, which means that vitamin C not directly protects cells but protects neurons by inhibiting the production of ROS.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.256

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