高糖对鹌鹑早期胚胎前肢NGF和BDNF的表达及神经形态发育的影响

High glucose inhibits the expression of NGF and BDNF in the forelimb of quail early embryos and the effect in developing peripheral nerve

目的:探讨高糖对鹌鹑早期胚胎前肢神经生长因子(NGF)和脑源性神经生长因子(BDNF)的表达及神经形态发育的影响。方法:受精日本鹌鹑蛋,随机分为对照组和实验组,对照组用0.7%氯化钠溶液处理,实验组分别用25、50和100 mmol/L浓度葡萄糖溶液处理,建立胚胎高糖发育模型,取胎龄E6.5阶段胚胎进行后续实验。通过检测顶臀径和体重探讨高糖对鹌鹑早期胚胎表型的影响;用免疫荧光染色法检测鹌鹑早期胚胎前肢神经的形态变化;反应性活性氧(ROS)检测试剂盒检测鹌鹑胚胎前肢活性氧水平;Western Blot检测鹌鹑胚胎前肢NGF和BDNF蛋白的表达变化;real time RT-PCR法检测NGF和BDNF的m RNA表达的变化。结果:鹌鹑早期胚胎表型测量显示,实验组的顶臀径和体重均有所下降(P <0.05),死亡率明显增加,部分胚胎表现出巨大胚胎和畸形发育体征;免疫荧光染色法检测结果表明,与对照组相比,各实验组鹌鹑早期胚胎前肢的beta Ⅲ Tubulin阳性神经纤维的积分光密度值(IOD)显著下降(P <0.05);活性氧含量检测显示,鹌鹑胚胎前肢ROS水平与对照组相比明显升高(P <0.05);real time RT-PCR检测各实验组NGF和BDNF m RNA的表达显著减少(P<0.05);Western Blot结果表明,NGF和BDNF蛋白的表达与对照组相比也显著减少(P <0.05)。结论:高糖引起鹌鹑早期胚胎前肢组织内高水平的氧化应激,而高水平的氧化应激可能影响了NGF和BDNF的表达,这或许是影响前肢神经形态发育的主要原因之一。

Objective: To investigate the effects of high glucose on the expression of nerve growth factor( NGF) and brain-derived nerve growth factor( BDNF) in quail early embryo and the development of forelimb nerve. Methods: Fertilized Japanese quail eggs were randomly divided into the control group and the experimental group. The control group was treated with 0. 7% sodium chloride solution. The experimental group was treated with 25,50,100 mmol/L high glucose solution respectively to establish high glucose developing embryos model and to take embryonic day 6. 5 embryo of quail for experiments. The effects of high glucose in physical developing quail embryos were explored by measuring the crown-rump length and body weight. Immunofluorescence staining for beta Ⅲ Tubulin was used to detect the morphology of nerve fibers. The content of reactive oxygen species( ROS) in the forelimbs of quail embryos were detected by ROS detection kit. The relative expressions of m RNA of NGF and BDNF in the forelimbs of quail embryos were detected by real time RT-PCR. The relative expressions of protein of NGF and BDNF in the forelimbs of quail embryos were performed by Western Blot. Results: Compared with the control group,the crown-rump length and body weight of the experimental group were decreased( P < 0. 05). The mortality rate increased significantly and some embryos showed signs of giant embryos and malformed embryos. Immunofluorescence staining shows that,compared with the control group,the IOD of beta Ⅲ Tubulin were significantly decreased( P < 0. 05). The content of ROS in the forelimb of quail embryo was significantly increased( P < 0. 05). The relative expression of m RNA of NGF and BDNF were significantly decreased( P < 0. 05). There was also a significant decrease in the expression of protein( P < 0. 05). Conclusion:High glucose induced high levels of ROS in the forelimb tissues of early quail embryos,and high levels of ROS may affect the expression of NGF and BDNF,which may be one of the main reasons affecting the morphological development of forelimb nerves.