环状RNA-ZNF292对缺氧缺血性脑损伤细胞氧化及凋亡的影响  

Effects of circular RNA-ZNF292 on cell oxidation and apoptosis in hypoxic-ischemic brain injury

作　　者：韩硕 徐涛[1] 赵亮[1] 戴大伟 张建忠 侯立军[1] HAN Shuo;XU Tao;ZHAO Liang;DAI Da-wei;ZHANG Jian-zhong;HOU Li-jun(Department of Neurosurgery,Changzheng Hospital,Naval Medical University(Second Military Medical Uinversity),Shanghai 200003,China)

机构地区：[1]海军军医大学(第二军医大学)长征医院神经外科,上海200003

出　　处：《第二军医大学学报》2021年第12期1341-1348,共8页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(81671206);上海市科学技术委员会国际科技合作项目(09410705100,14430721300);军队“十二五”重点项目(BWS12J025).

摘　　要：目的建立原代神经元缺氧缺血性脑损伤细胞模型,探讨环状RNA-ZNF292(cZNF292)对脑缺血损伤后神经元氧化应激及凋亡的影响。方法选取胎龄为18 d的胎鼠培养原代神经元,采用4、10 mmol/L Na_(2)S_(2)O_(4)和无糖培养基处理细胞1、2、3、4 h后复氧培养0、5、15、30 h,建立氧糖剥夺/复氧细胞模型。通过细胞骨架蛋白和β3-微管蛋白免疫荧光染色观察原代神经元的形态变化,FITC标记鬼笔环肽染色观察不同浓度Na_(2)S_(2)O_(4)对原代神经元存活率的影响。采用ELISA检测培养上清液中活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)和乳酸脱氢酶(LDH)的表达水平。采用蛋白质印迹法检测细胞中caspase 3及细胞周期蛋白G1(CCNG1)的表达水平。结果成功建立缺氧缺血性脑损伤细胞模型和cZNF292敲减细胞模型,并发现缺氧培养4 h时复氧、10 mmol/L的Na_(2)S_(2)O_(4)对原代神经元的生长抑制率最高,且在复氧培养15 h时细胞中cZNF292表达水平最高,因此选择缺氧培养4 h、复氧培养15 h、10 mmol/L Na_(2)S_(2)O_(4)为最佳实验条件。氧糖剥夺使细胞培养基中ROS和MDA水平上升,SOD和LDH水平下降(P均<0.05);而敲减cZNF292后,细胞培养基中ROS和MDA水平下降,SOD和LDH水平上升(P均<0.05)。敲减cZNF292可使caspase 3和CCNG1表达下降(P均<0.05)。结论脑缺血可以诱导原代神经元cZNF292表达增加,而敲减cZNF292可以缓解原代神经元在缺血缺氧环境下发生的氧化损伤、抑制细胞凋亡及增殖。Objective To establish a cell model of primary neuronal hypoxic-ischemic brain injury and explore the effects of circular RNA-ZNF292(cZNF292)on neuronal oxidative stress and apoptosis after hypoxic-ischemic brain injury.Methods The primary neurons were cultured in fetal rats on the 18^(th) day of pregnancy.The cells were treated with 4,10 mmol/L Na_(2)S_(2)O_(4)and glucose-free medium for 1,2,3 and 4 h,and reoxygenated for 0,5,15 and 30 h to establish the oxygen and glucose deprivation/reoxygenation cell model.The morphological changes of primary neurons were observed by immunofluorescence staining of cytoskeletal proteins andβ3-tubulin,the effects of different concentrations of Na_(2)S_(2)O_(4)on the survival rate of primary neurons were observed by fluorescein isothiocyanate(FITC)-phalloidin staining.The expression levels of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD)and lactate dehydrogenase(LDH)in the culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of caspase 3 and cyclin G1(CCNG1)were detected by Western blotting.Results The hypoxic-ischemic brain injury cell model and cZNF292-knockdown model were successfully established and the highest inhibition rate was observed in 10 mmol/L Na_(2)S_(2)O_(4)and reoxygenation 4 h after deoxygenation.The expression level of cZNF292 was the highest in cells after reoxygenation culture for 15 h.Therefore,hypoxia culture for 4 h,reoxygenation culture for 15 h and 10 mmol/L Na_(2)S_(2)O_(4)were selected as the best experimental conditions.Oxygen and glucose deprivation increased the levels of ROS and MDA,and decreased the levels of SOD and LDH in cell culture medium(all P<0.05);after knockdown of cZNF292,the levels of ROS and MDA were decreased,and the levels of SOD and LDH were increased(all P<0.05).Knockdown of cZNF292 decreased the expression of caspase 3 and CCNG1(both P<0.05).Conclusion Cerebral ischemia can increase expression of cZNF292 in primary neurons,while knockdown of cZNF292 can

关 键 词：环状RNA 锌指蛋白292 氧糖剥夺/复氧 细胞模型 脑缺血 氧化损伤 细胞凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]