低温对小鼠N2a细胞氧糖剥夺/复氧复糖时内质网-线粒体结构偶联的影响  

Effect of hypothermia on mitochondria-associated endoplasmic reticulum membranes during oxygen-glucose deprivation and reoxygenation in mouse N2a cells

作　　者：陈怀龙[1] 张艳平 于群 王炳琪 高翔[3] 张高峰[1] 时飞[1] Chen Huailong;Zhang Yanping;Yu Qun;Wang Bingqi;Gao Xiang;Zhang Gaofeng;Shi Fei(Department of Anesthesiology,Qingdao Municipal Hospital,Qingdao 266071,China;Department of Anesthesiology,Weifang Medical College,Weifang 261053,China;Department of Clinical Medicine,Weifang Medical College,Weifang 261053,China)

机构地区：[1]青岛市市立医院麻醉科,266071 [2]潍坊医学院麻醉学院,261053 [3]潍坊医学院临床学院,261053

出　　处：《中华麻醉学杂志》2020年第11期1358-1361,共4页Chinese Journal of Anesthesiology

基　　金：青岛市民生科技计划项目(19-6-1-50-nsh)。

摘　　要：目的评价低温对小鼠N2a细胞氧糖剥夺/复氧复糖时内质网-线粒体结构偶联(MAMs)的影响。方法将生长状态良好的小鼠N2a细胞采用随机数字表法分为5组(n=24):对照组(C组)、氧糖剥夺/复氧复糖组(OGD/R组)、低温组(H组)、siRNA-Mfn2转染+低温组(siMfn2+H组)和siRNA-NC转染+低温组(siNC+H组)。C组在正常条件下培养;OGD/R组氧糖剥夺3 h后复氧复糖24 h;H组氧糖剥夺3 h后,在32 ℃低温下复氧复糖24 h;siMfn2+H组和siNC+H组模型制备前48 h时分别转染siRNA-Mfn2及非特异性siRNA,余同H组。于复氧复糖24 h时,采用CCK-8法检测细胞存活率,qRT-PCR法检测Mfn2 mRNA表达水平,Western blot检测Mfn2表达水平,透射电镜下观察并测量内质网-线粒体偶联部分长度、内质网周长和线粒体周长,计算内质网-线粒体偶联部分长度/内质网周长比值和内质网-线粒体偶联部分长度/线粒体周长比值,反映MAMs水平。结果与C组比较,其余4组细胞存活率、Mfn2及其mRNA表达水平和MAMs水平降低(P<0.05);与OGD/R组比较,H组和siNC+H组细胞存活率、Mfn2及其mRNA表达水平和MAMs水平升高(P<0.05),siMfn2+H组上述指标差异无统计学意义(P>0.05);与H组比较,siMfn2+H组细胞存活率、Mfn2及其mRNA表达水平和MAMs水平降低(P<0.05),siNC+H组上述指标差异无统计学意义(P>0.05)。结论低温减轻小鼠N2a细胞氧糖剥夺/复氧复糖损伤的机制与上调Mfn2表达,从而稳定MAMs有关。Objective To evaluate the effect of hypothermia on the mitochondria-associated endoplasmic reticulum membranes(MAMs)during oxygen-glucose deprivation and reoxygenation(OGD/R)in the mouse neuroblastoma cells(N2a).Methods The well-growing N2a cells were divided into 5 groups(n=24 each)using a random number table method:control group(group C),group OGD/R,hypothermia group(group H),transfection with siRNA-Mfn2 plus hypothermia group(group siMfn2+H),and transfection with siRNA-NC plus hypothermia group(group siNC+H).The cells were cultured in normal condition in group C.The cells were subjected to O2-glucose deprivation(OGD)for 3 h followed by restoration of O2-glucose supply for 24 h in group OGD/R.The cells were subjected to OGD for 3 h followed by restoration of O2-glucose supply for 24 h at 32℃in group H.In siMfn2+H and siNC+H groups,cells were transfected with siMfn2 and non-specific interfering RNA,respectively,at 48 h before establishing OGD/R model,and the other treatments were similar to those previously described in group H.At 24 h of reoxygenation,cell survival rate was recorded by the cell counting kit-8 assay,the expression of mitofusin 2(Mfn2)mRNA and protein was detected by quantitative real-time-polymerase chain reaction or Western blot,and the length of the coupling structure,endoplasmic reticulum perimeter and mitochondrial perimeter were observed and measured with a transmission electron microscope.The ratio of the length of the coupling structure/endoplasmic reticulum perimeter and ratio of the length of the coupling structure/mitochondrial perimeter were calculated to reflect the level of MAMs.Results Compared with group C,the cell survival rate and level of MAMs were significantly decreased,and the expression of Mfn2 protein and mRNA was down-regulated in the other four groups(P<0.05).Compared with group OGD/R,the cell survival rate and level of MAMs were significantly increased,and the expression of Mfn2 protein and mRNA was up-regulated in H and siNC+H groups(P<0.05),and no significant change

关 键 词：低温 人工 再灌注损伤 脑 内质网-线粒体结构偶联 

分 类 号：R614[医药卫生—麻醉学]