【作者】 刘晓艳；

【导师】 徐剑文； 王玮；

【作者基本信息】 福建医科大学 ， 神经生物学， 2009， 硕士

【摘要】 目的观察短暂性缺氧后鼠脑神经源性分化因子(Neurogenic differentiation, NeuroD)表达量的变化及其与神经系统再生的关系,探讨其在神经系统再生中的可能作用。方法通过RT-PCR检测短暂性缺氧后NeuroD mRNA表达量的变化。进而通过体外培养大鼠海马神经元,RT-PCR检测细胞缺氧后NeuroD mRNA表达量的变化,BrdU免疫组化检测缺氧后细胞增殖情况;通过“延迟剖宫产术”建立胎鼠宫内窘迫模型,对模型组动物进行神经行为学检测,原位杂交技术检测术后不同天数海马结构NeuroD mRNA表达量的变化。结果短暂性缺氧后NeuroD mRNA表达量增高,并且表达量随着缺氧时间的延长而不断增高。免疫荧光结果显示,在大脑皮质及皮质下区域NeuroD呈点状或条索状弥散分布,窘迫前后NeuroD表达量未见明显变化(P>0.05)。在尾壳核区域NeuroD的表达相对集中,窘迫后表达量明显增高。体外培养5d的海马神经元缺氧3h后RT-PCR检测到NeuroD mRNA表达量明显增高,BrdU免疫组化结果显示缺氧再培养后检测到BrdU阳性细胞,即有的神经元返回细胞周期,重新进行分裂增殖。在体通过“延迟剖宫产术”建立宫内窘迫模型。大鼠神经行为学检测结果表明,宫内窘迫10min组和对照组相比,大鼠的动作协调能力和学习能力统计学上未见明显差异,但模型组大鼠的重拾记忆能力有明显提高(P< 0.05)。原位杂交结果显示,模型组无论在海马结构的齿状回颗粒下层还是CA1区,NeuroD在P13、P20、P27天的表达量与对照组相比都有明显增高,且在P20天最明显。结论1.在体动物实验证明短暂性缺氧后NeuroD表达量增高2.体外培养海马神经元缺氧后NeuroD表达量增高,与之伴随的是有的神经元返回细胞周期,重新进行分裂增殖3.海马齿状回颗粒下层和CA1区NeuroD表达量的变化与神经系统再生具有相关性以上表明,高表达的NeuroD可能参与了神经干细胞的分化,在神经系统再生过程中发挥了一定作用更多还原

【Abstract】 Objective To observe the influence of transient hypoxia on the expression of NeuroD in rat brain and investigate its possible roles in neural regeneration.Methods To investigate the influence of transient hypoxia on the expression of NeuroD, asphyxia was induced in rat pups by performing a delayed cesarean section on pregnant Sprague-Dawley rats.The expression of NeuroD was measured by RT-PCR and immunofluorescence. To investigate the role of NeuroD in neural regeneration, the delayed cesarean section was performed on pregnant SD rats and the newborn rats were returned to dams. In vitro, influence of transient hypoxia on the expression of NeuroD was analyzed on the outcome of embryonic rat neurons in culture. The expression of NeuroD was measured by RT-PCR and the cell proliferation was measured by incorporation of BrdU. In vivo, neurobehavioral consequences of the hypoxic episode were evaluated by using standard behavioral tests for psychomotor and coordination performances and learning capacities.The expression of NeuroD was monitored at the level of DG and CA1 layer of the hippocampus by in situ hybridization.Results NeuroD mRNA increased after the exposure to hypoxia ( P < 0.05 ).Compared with the control group, NeuroD showed no much difference at cerebral cortex but increased significantly at nucleus caudatus putamen detected by immunofluorescence. As revealed by neurobehavioral consequences, hypoxia did not impair psychomotor or learning capacities but improved memory retrieval scores. In vitro, following hypoxia for 3 hours in cultured neurons, NeuroD increased distinctly and with incorporation of BrdU revealed an accumulation of proliferating cells. Following hypoxia in vivo induced higher expression of NeuroD in DG and CA1 subfield of the hippocampus at days 13、20、27 post hypoxia and significantly at P20. Conclusions First, the expression of NeuroD was increased the exposure to hypoxia. Second, the expression of NeuroD was increased post asphyxia in cultured neurons, following with cell proliferation. Third, the differential expression of NeuroD coincided with the dalyed brain neurogenesis. As mentioned above, NeuroD seems to play a role in the process of neurogenesis.更多还原