Objective To explore the effects of different severities of neuronal injury on microglial phenotype. Methods Primary neurons were treated by hypoxia for 0.5 ,1 ,2 ,and 4h,and then reoxygenated for 24 h.Neuron‐conditioned media (NCM)were collected and added to microglial cultures(1∶1 volume).Twenty‐four h later ,Western blot was used to detect the expressions of the microglial M 2 phenotype marker arginase‐1 and the activation marker ionized calcium binding adapter mole‐cule 1(Iba‐1).Immunofluorescence was performed to detect the expression of arginase‐1.Additionally ,microglial trophic mole‐cules brain‐derived neurotrophic factor(BDNF) ,glial cell line‐derived neurotrophic factor(GDNF)and proinflammatory cyto‐kines TNF‐α,IL‐1β,IL‐6 were determined by ELISA. Finally ,different phenotypes of primary microglia were formed after cells were treated with different NCM.MTT assays were used to determine the viability of neurons which were treated by hypoxia for 2 h and then co‐cultured with microglia for 24 h.Results NCM from mild injuries(treated by hypoxia for 0.5 or 1 h)signifi‐cantly down‐regulated the expression of the microglial M 2 phenotype marker arginase‐1 ,and those from moderate and severe in‐juries up‐regulated the arginase‐1 expression. Iba‐1 was upregulated in all injury conditions ,suggesting that all the injury condi‐tions can induce microglial activation to some degree.Moreover ,NCM from mild injuries up‐regulated the secretion of microglial TNF‐α,IL‐1βand IL‐6 ,but NCM from moderate(hypoxia 2 h)and severe(hypoxia 4 h)injuries had no effects on these proin‐flammatory cytokines. NCM of different injury severities significantly up‐regulated the release of these trophic molecules. MTT assays demonstrated that microglia incubated with NCM of mild and severe injuries further exacerbated the injury of neurons treated by hypoxia.Microglia incubated with NCM of moderate injuries protected neurons from hypoxia injury.Conclusion The severity of neuronal injury is an important factor in determining microglial phenotypes ,which has neurotoxic or neuroprotective effects.%目的：探讨不同的神经元损伤度对小胶质细胞表型的影响。方法将原代培养的神经元进行缺氧处理，不同的时长（0.5、1、2、4 h）后，复氧处理24 h ，收集神经元条件培养液（neuron‐conditioned media ，NCM ），然后将NCM刺激原代培养的小胶质细胞24 h（NCM∶小胶质细胞培养液＝1∶1，V/V ）。采用Western blot法检测小胶质细胞内的M2表型标记物精氨酸酶‐1（arginase‐1）和激活标记物离子钙结合衔接分子‐1（Iba‐1）的表达变化规律，采用免疫荧光法检测ar‐ginase‐1的表达变化规律。同时，采用 ELISA 法检测小胶质细胞培养液上清中的营养因子，如脑源性神经营养因子（BDNF）、胶质细胞源性神经营养因子（GDNF）和炎性因子，如肿瘤坏死因子‐α（TNF‐α）、白细胞介素‐1β（IL‐1β）、白细胞介素‐6（IL‐6）的分泌变化规律。最后，将不同损伤度的NCM 诱导小胶质细胞形成不同表型，将小胶质细胞和缺氧处理2 h后的神经元共培养24 h ，MTT法检测神经元的活力。结果轻微损伤（缺氧0.5、1 h）的NCM 明显下调M2型标记物arginase‐1的表达水平，中度（缺氧2 h）和重度（缺氧4 h）损伤的 NCM 能够上调arginase‐1的表达，各种损伤度的NCM都能上调Iba‐1的表达水平，提示其在一定程度上激活小胶质细胞。同时，轻微损伤的NCM明显上调小胶质细胞TNF‐α、IL‐1β和IL‐6的分泌，而中度和重度损伤的NCM 对这些促炎因子的释放没有影响，各种损伤度的NCM 都能明显上调营养因子的分泌。M T T法检测表明，轻微和重度损伤处理的NCM刺激的小胶质细胞进一步加重缺氧处理的神经元损伤，而中度损伤的NCM对缺氧处理后的神经元具有保护作用。结论神经元损伤度是决定小胶质细胞表型的重要因素，进而使小胶质细胞进一步发挥神经毒性或保护作用。
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