裸鼹鼠海马神经元纯化模型建立及其低氧耐受特性机制的初步研究

Establishment of a Purified Hippocampal Neuron Model in Naked Mole-rat and Preliminary Study on Its Hypoxic Tolerance

目的建立一种裸鼹鼠海马神经元的纯化培养方法,并初步探讨其低氧耐受特性。方法收集妊娠60~65 d裸鼹鼠胚胎海马组织内的细胞,利用含有Neurobasal+2%B27+10μmol/L5-氟尿嘧啶的培养基进行神经元纯化,海马神经元维持培养基为含有体积分数2%B27的Neurobasal培养基。体外贴壁培养6~7 d,采用倒置显微镜观察细胞的生长状态,利用神经元特异核蛋白(NeuN)抗体进行鉴定。利用CCK8试剂盒进行海马神经元低氧耐受功能的研究(低氧浓度为8%)。利用QPCR检测海马神经元中缺氧诱导因子-1α(HIF-1α)在低氧情况下的表达水平。结果通过本方法获得的海马神经元具有典型的神经元形态特征,免疫细胞化学染色显示纯化所得的细胞均呈NeuN阳性,低氧条件下细胞存活率、轴突长度显著大于常氧情况下的数值,低氧情况下裸鼹鼠海马神经元中HIF1α显著高于常氧情况,上述结果差异均具有统计学意义(P<0.05)。结论建立了一种简单实用的体外分离培养裸鼹鼠海马神经元的方法,初步揭示了裸鼹鼠海马神经元低氧耐受特性,并且该特性与裸鼹鼠海马神经元于低氧情况下高表达HIF1α有关。

Objective To establish a method for purifying and culturing hippocampal neurons of naked mole-rats and to preliminarily explore their hypoxic tolerance characteristics. Methods Cells in hippocampus of naked mole-rats during 60-65 days of gestation were collected, and purified by the medium containing Neurobasal+2%B27+10 μmol/L 5-fluorouracil. Hippocampal neurons were maintained by neurobasal medium containing 2% B27. Cell growth was observed by inverted microscope after 6-7 days of adherent culture in vitro. Neuron-specific antibody was used to identify neuronspecific antibody. CCK8 kits were used to study on hypoxic tolerance of hippocampal neurons.Axonal length of naked mole-rat hippocampal neurons were measured by Image-Pro Plus 6.0. QPCR analysis was used to detect the hypoxia-inducible factor 1α(HIF1α) mRNA levels of hippocampal neurons under normoxia and hypoxia conditions. Results The hippocampal neurons obtained by this method exibits typical morphological characteristics of neurons, and immunocytochemical staining shows that the purified cells are NeuN-positive. The cell survival rate under hypoxia is more than twice of that under conditions with normal oxygen, and the axon lengths of cells in hypoxia conditions were smaller than those under nomoxia conditions. HIF1α levels of naked mole-rat hippocampal neurons were higher than those under normoxia conditions. There are statistically differences among these results(P<0.05). Conclusion The method established in this study is a simple and practical method for isolating and culturing hippocampal neurons of naked mole-rats in vitro, and has strong hypoxic tolerance.