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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 changes of neurogenic differentiation factor (NeuroD) expression after transient hypoxia and explore its possible role in the nervous system regeneration. Methods Hippocampal neurons were cultured in vitro and identified by neuron-specific enolase (NSE) immunohistochemistry and Nissl staining. Neurons cultured for 5 days were cultured hypoxic in a three-gas incubator (37 ° C, 94% N2, 5% CO2, 1% O2). The expression of NeuroD mRNA in neurons was detected by RT-PCR 3 h and 6 h after hypoxia. Morphological changes of neurons were observed by electron microscope. Neurons exposed to hypoxia for 3 hours were cultured in a 5% CO 2 incubator at 37 ℃ for 96h, and BrdU (final concentration was 10μmol / L) was added 48h before fixation. Immunohistochemistry With or without cell proliferation. Results RT-PCR showed that NeuroD expression was significantly increased 3 h after hypoxia (P <0.01), but no significant difference was found between NeuroD expression and control group 6 h after hypoxia (P> 0.05). Electron microscopy results showed that after 3 hours of hypoxia, no significant changes were found in intracellular organelles. After 6 hours of hypoxia, vacuoles were observed in the cells and mitochondria swell obviously. BrdU positive cells were detected by immunohistochemistry. Conclusion The expression of NeuroD is increased after transient hypoxia, which may be involved in the regeneration of the nervous system.