短暂性缺氧后海马神经元神经源性分化因子表达增高

Expression of neurogenic differentiation factor increased after transient hypoxia of hippocampal neurons in vitro

目的观察短暂性缺氧后神经源性分化因子(NeuroD)表达量的变化,探讨其在神经系统再生中的可能作用。方法体外培养海马神经元,经神经元特异性烯醇化酶(NSE)免疫组织化学方法、尼氏体染色法鉴定。将培养5d的神经元置于三气培养箱(37℃、94%N2、5%CO2、1%O2)内缺氧培养。RT-PCR检测缺氧3h和6h后神经元NeuroD mRNA表达水平,电镜观察神经元形态学变化。将缺氧3h的神经元置于37℃、5%CO2培养箱中继续培养96h,固定前48h加入5-溴脱氧尿嘧啶核苷(BrdU)(终浓度为10μmol/L),免疫组织化学检测有无细胞增殖。结果 RT-PCR显示,缺氧3h后NeuroD表达量明显增高(P<0.01),而缺氧6h后NeuroD表达量与对照组相比差异无统计学意义(P>0.05)。电镜结果显示,缺氧3h后细胞内细胞器未见明显变化,缺氧6h后细胞内出现空泡样变化,线粒体肿胀明显。免疫组织化学检测到BrdU阳性细胞。结论短暂性缺氧后NeuroD表达量增高,可能参与了神经系统的再生过程。

Objective To observe the influence of transient hypoxia on the expression of neurogenic differentiation factor（NeuroD） in cultured neurons and investigate its possible roles in neural regeneration. Methods Influence of transient hypoxia on the expression of NeuroD was analyzed on the outcome of embryonic rat neurons in culture. Cultures at five days were exposed to hypoxia. After different periods（3 hours, 6 hours） of hypoxia, RT-PCR was performed to examine the mRNA levels of NeuroD. Electron microscopy was performed to observe neuronal alterations. Dishes after 3 hours hypoxia were then returned to normal atmosphere for ensuing culture 96 hours. Immunohistoehemistry was performed to examine cell proliferation by incorporation of 5-bromodeoxyuridine（ BrdU）. Results Following hypoxia for 3 hours in cultured neurons, NeuroD increased distinctly and the incorporation of BrdU revealed an accumulation of proliferating cells. Compared with the control group, NeuroD showed no much difference after hypoxia for 6 hours （ P 〉 0. 05）. Neurons exposing hypoxia for 6 hours were damaged by electron microscope. Conclusion The expression of NeuroD increases post asphyxia in cultured neurons, following with cell proliferation. NeuroD seems to play a role in the process of neurogenesis.