不同类型神经细胞对低氧的敏感性研究

The Sensitivity of Different Neural Cells to Hypoxia

目的:阐明低氧对神经细胞在能量代谢、氧化应激、炎症等方面的影响,探讨不同类型神经细胞响应低氧刺激的敏感性,为低氧诱导的脑损伤病理机制研究提供理论基础。方法:将不同类型的神经细胞系(BV2、N9、Gl261、HT22)低氧(0.1%O_(2),5%CO_(2))处理0~24 h,用CCK8法(Cell Counting Kit-8)检测细胞增殖,发光细胞活力测定法(luminescent cell viability assay)检测细胞活力。提取各细胞总RNA及总蛋白质,通过实时荧光定量PCR(quantitative real time-PCR,RT-qPCR)、免疫印迹(Western blot)、流式细胞仪等技术检测能量代谢、炎症相关基因表达水平和氧化应激水平。结果:低氧处理后,各类神经细胞增殖明显减慢。小胶质细胞BV2和N9对低氧的响应主要表现在炎症因子和糖代谢相关酶的改变,星形胶质细胞Gl261和神经元HT22对低氧的响应主要表现在糖代谢相关酶的改变,小胶质细胞和星形胶质细胞在低氧刺激下氧化应激水平显著增加。结论:不同类型神经细胞响应低氧刺激的敏感性不同。能量代谢和炎症反应方面,小胶质细胞对低氧处理更敏感,表现为糖酵解酶显著上调,炎症因子变化明显;氧化应激方面,小胶质细胞和星形胶质细胞对低氧处理更敏感,表现为氧化应激响应快,ROS产生显著增加。

Objective:In order to illustrate the hypoxia-induced changes of neural cells in inflammatory response,oxidative stress,and energy metabolism process and to compare the sensitivity of neural cells’responses to hypoxia.Methods:Different types of neural cells(BV2,N9,Gl261,HT22)were treated with hypoxia(0.1%O_(2),5%CO_(2))for 0-24 hours.Cell proliferation was detected by Cell Counting Kit-8 method and cell viability was assayed by CellTiter-Glo Luminescent Cell Viability Assay.Total RNA was extracted by Trizol reagent,and the inflammation,oxidative stress,and energy metabolism-related genes expression were measured by quantitative real-time PCR and Western blot.The ROS production was detected by flow cytometer with fluorescence probe.Results:Hypoxia stimulation decreased cell proliferation and cell viability.The hypoxia-induced changes of microglial cells(BV2 and N9)were mainly involved in inflammatory response and glucose metabolism process.The changes of astrocytes Gl261 and neural cell HT22 were mainly involved in glucose metabolism process.Hypoxia stimulation significantly increased oxidative stress in microglia and astrocytes.Conclusion:Different types of neural cells have different degrees of sensitivity in response to hypoxic stimulation.In terms of energy metabolism and inflammatory response,microglia are more sensitive to hypoxia treatment,which is manifested as a significant up-regulation of glycolytic enzymes and inflammation genes,whereas microglia and astrocytes are more sensitive to hypoxia treatment in terms of oxidative stress,which is indicated by their quick response and significant increase of ROS production.