解耦联蛋白2对氧糖剥夺/再复供神经细胞的保护作用及机制研究  

Protective effect and mechanism of uncoupling protein 2 on oxygen-glucose deprivation/resupply neurons

作　　者：王天宇 陈秋月 陈琪 张叶青 陈富强 陈云 Wang Tianyu;Chen Qiuyue;Chen Qi;Zhang Yeqing;Chen Fuqiang;Chen Yun(Department of Neurology,2Department of Precision Medicine Center,Taizhou Central Hospital(Taizhou University Affiliated Hospital),Taizhou 318000,Zhejiang,China)

机构地区：[1]台州市中心医院(台州学院附属医院)神经内科,浙江台州318000 [2]台州市中心医院(台州学院附属医院)精准医学中心,浙江台州318000

出　　处：《中国中西医结合急救杂志》2022年第6期680-684,共5页Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care

基　　金：浙江省医药卫生科技计划项目(2019KY789);浙江省台州市科技计划项目(20ywb50)

摘　　要：目的探讨解耦联蛋白2(UCP2)对氧糖剥夺/再复供(OGD/R)神经细胞的保护作用及其机制。方法原代培养小鼠海马神经元细胞,融合率达80%左右可进行传代培养。取对数期细胞,采用UCP2-干扰小RNA(siRNA)转染的神经细胞沉默UCP2表达;将转染后的细胞随机分为UCP2-siRNA组和阴性对照siRNA组(NC-siRNA组)。采用神经元特异性烯醇化酶(NSE)免疫荧光染色鉴定细胞,倒置相差显微镜下观察细胞状态,细胞增殖采用四甲基偶氮唑盐法(MTT)检测。用转染后的细胞制备OGD/R模型。两组分别于复氧复糖后即刻和12、24、48 h收集细胞,应用相应试剂盒检测细胞内三磷酸腺苷(ATP)、活性氧(ROS)、琥珀酸脱氢酶(SDH)的表达水平。结果倒置相差显微镜下观察显示,NC-siRNA组细胞饱满,折光性强,胞核明显,突起清晰;海马神经元细胞经UCP2-siRNA转染干扰沉默UCP2后,细胞皱缩,折光性差,核质不清,突起不清晰。MTT法检测细胞增殖情况提示,两组细胞增殖水平均随接种时间延长呈升高趋势,从接种后48 h开始,UCP2-siRNA组各时间点细胞增殖水平均明显低于NC-siRNA组,以接种96 h时最为明显〔吸光度(A)值:1.617±0.008比1.828±0.048,P<0.05〕。随复氧复糖时间的延长,两组细胞ATP和SDH表达均呈下降趋势,ROS表达呈升高趋势,其中UCP2-siRNA组复氧复糖后即刻及12、24、48 h的ATP和SDH表达水平均明显低于NC-siRNA组,ROS表达水平均明显高于NC-siRNA组,以复氧复糖48 h时最为明显〔ATP(RLU):0.33±0.09比0.50±0.11,SDH(U/mg):0.30±0.08比0.57±0.08,ROS(μmol/L):194.54±6.64比177.29±9.35,均P<0.05〕。结论UCP2对OGD/R神经细胞存在保护作用,UCP2可能通过改善能量代谢、抗氧化等方面达到对OGD/R神经细胞的保护作用。Objective To explore the protective effect and mechanismof uncoupling protein 2(UCP2)on oxygen-glucose deprivation/resupply(OGD/R)neurons.Methods The primary hippocampal neurons of mice were sub-cultured when the cell fusion reached about 80%.The neurons were transfected with UCP2-small interfering RNA(siRNA)to silence the expression of UCP2.After transfection,the cells were randomly divided into UCP2-siRNA group and normal control siRNA(NC-siRNA)group.Neuron specific enolase(NSE)immunofluorescence staining was used for identifying characteristics of the cells under light microscope.Cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)assay.The transfected cells were used to prepare the OGD/R model.Cells in the two groups were collected at 0,12,24,and 48 hours after restoration of oxygen and glucose,respectively.The expression levels of adenosine triphosphate(ATP),reactive oxygen species(ROS),and succinate dehydrogenase(SDH)in cells were detected with corresponding kits.Results Under the inverted phase contrast microscope,the cells in the NC-siRNA group were plump and refractive,with obvious nucleus and clear synapses.The cells in the UCP2-siRNA were shrunk,with poor refractive index,whose nucleus and synapses were unclear.MTT assay showed that the cell proliferation levels of the two groups increased with the extension of inoculation time.From 48 hours after inoculation,the cell proliferation level of UCP2-siRNA group was significantly lower than that of NC-siRNA group at each time point,especially at 96 hours[absorbance(A)value:1.617±0.008 vs.1.828±0.048,P<0.05].With the prolongation of resupply,the expression of ATP and SDH in the two groups showed a downward trend,and the expression of ROS showed an increasing trend.The expression levels of ATP and SDH in UCP2-siRNA group were significantly lower than those in NC-siRNA group at 0,12,24 and 48 hours after resupply.ROS expression levels were significantly higher than those in NC-siRNA group.The above differences were most obvious at 48 hours af

关 键 词：解耦联蛋白2 氧糖剥夺/再复供 神经细胞 保护作用 

分 类 号：R73[医药卫生—肿瘤]