地塞米松通过下调Reelin表达抑制小鼠胚胎大脑皮质神经元迁移

Dexamethasone Inhibits Neuronal Migration in Mouse Fetal Cerebral Cortex by Down-regulating Reelin Expression

为探究小鼠孕晚期地塞米松应用对胚胎大脑皮质神经元迁移以及大脑发育的影响,健康孕鼠12只被随机分为试验组和对照组,试验组于母鼠孕第14.5~18.5天腹腔注射0.4 mg/kg地塞米松;对照组给予等剂量生理盐水。仔鼠于P0被断头取脑,多聚甲醛固定后振荡切片,免疫荧光标记大脑皮层Brn2阳性神经元,激光共聚焦显微镜拍照,观察皮层神经元迁移情况;仔鼠P4冰冻麻醉,大脑皮质蛋白取材,蛋白印迹检测仔鼠大脑皮质中摇晃蛋白(Reelin)的表达量。结果显示,对照组仔鼠脑重177.76 mg±3.79 mg,而试验组仔鼠脑重168.80 mg±4.24 mg,地塞米松处理后仔鼠大脑重量减轻;免疫荧光染色结果显示,对照组P0仔鼠中,由Brn2阳性神经元组成的大脑皮质Ⅱ-Ⅲ层的深度为122.06μm±5.37μm,试验组深度为158.20μm±9.54μm,处理组较对照组明显加深;蛋白印迹检测结果表明,地塞米松处理组P4仔鼠大脑皮质中Reelin表达下降。结果证明,孕晚期应用地塞米松可引起胚胎大脑皮质Reelin表达减少和神经元迁移障碍,导致大脑发育迟缓。

To explore the effects of dexamethasone application in late pregnancy on cerebral cortical neuronal migration and brain development in embryonic mice,12 pregnant mice were randomly divided into dexamethasone group and control group.The dexamethasone group was intraperitoneally injected with dexamethasone at a daily dose of 0.4 mg/kg body weight from embryonic day(E)14.5 to 18.5,the control group was given an equal dose of saline.For the preparation of brain slices,the offspring were sacrificed on postnatal day(P0).Brains were gathered,fixed in 4%paraformaldehyde(PFA)and sliced with a vibratome.Sections were then immunofluorescence stained with Brn2 antibody to label the late-born neurons inⅡ-Ⅲcortical layers.Images were acquired using a Laica SP8 confocal microscopy.Western blotting was used to detect the expression of Reelin protein in the cerebral cortex of the offspring.The results showed that the brain weight of the offspring was reduced after dexamethasone treatment:the control group weighed 177.76 mg±3.79 mg,while the dexamethasone group weighed 168.80 mg±4.24 mg.Immunofluorescence staining results showed that the depth ofⅡ-Ⅲlayers in the cerebral cortex composed of Brn2 positive neurons was 122.06μm±5.37μm in the control group,while the depth of corticalⅡ-Ⅲlayers in the dexamethasone group was 158.20μm±9.54μm,the depth of dexamethasone group was much deeper than that in the control group.Western blotting analysis indicated that the expression of Reelin protein in the cerebral cortex treated with dexamethasone is significantly reduced.Therefore,our results prove that the application of dexamethasone during pregnancy can cause decrease of Reelin expression and migration defect of neurons in the embryonic cerebral cortex,and result in brain development retardation.