内质网应激在高糖培养加重神经细胞缺氧-复氧损伤中的作用

Effect of endoplasmic reticulum stress in high glucose aggravating hypoxia-reoxygenation injury in neuroblastoma cells

目的研究内质网应激在高糖培养加重神经细胞缺氧-复氧损伤中的作用。方法在含10%胎牛血清的DMEM/F12培养基中培养小鼠神经瘤母细胞N2a,按照接种密度每毫升105个将细胞接种于培养板中。采用随机数字分组法将细胞分为四组:正常糖对照组(NC组)、正常糖缺氧-复氧组(NH组)、高糖对照组(HC组)、高糖缺氧-复氧组(HH组)。待细胞贴壁后,将DMEM/F12培养基更换为高糖培养基,孵育48h,造高糖孵育模型。在缺氧小室中缺氧3h后,再转入正常氧孵育箱中复氧2h,造缺氧-复氧损伤模型。NC组未作处理;NH组行缺氧-复氧处理;HC组行高糖孵育处理;HH组先行高糖孵育48h后,再作缺氧-复氧处理。采用CCK-8法检测细胞活力,流式细胞仪检测细胞凋亡率,Western blot法检测细胞GRP78、CHOP蛋白含量。结果与NC组和HC组比较,NH组和HH组细胞活力明显减弱,凋亡率明显升高,GRP78、CHOP蛋白含量明显增多(P<0.01);与NH组比较,HH组细胞活力明显减弱,凋亡率明显升高,GRP78、CHOP蛋白含量明显增多(P<0.05)。结论高糖培养加重神经细胞缺氧-复氧损伤,可能与内质网应激标志性蛋白GRP78蛋白含量增多和促凋亡转录因子CHOP介导的细胞凋亡相关。

Objective To identify the effect of endoplasmic reticulum stress in high glucose aggravating hypoxia-reoxygenation injury in neuroblastoma cells.Methods Mouse neuroblastoma cells（N2 a）were cultured in DMEM/F12 culture medium supplemented with 10%fetal bovine serum.The cells were then divided into 4 groups using a random number table：normal glucose control group（group NC）,normal glucose hypoxia-reoxygenation group（group NH）,high glucose control group（group HC）,high glucose hypoxia/reoxygenation group（group HH）.The cells were incubated in high glucose culture medium（30.0 mmol/L）for 48 hto simulate high glucose damage model.To mimic the hypoxia-reoxygenation injury,the cells were incubated in a hypoxic chamber（under 95%nitrogen and 5% CO2）for 3 h,then transferred to a normoxic incubator（under 95% air and 5%CO2）for 2 hto reoxygenation.The cells in group NC were cultured without any treatment.The cells in group NH were subjected to hypoxia-reoxygenation injury only.The cells in group HC were cultured in high glucose medium.The cells in group HH were cultured with high glucose medium,and then subjected to hypoxia-reoxygenation injury.The cell viability was measured by CCK-8 assay,the cell apoptosis was detected by flow cytometry and the apoptosis rates were calculated.In addition,the contents of GRP78,CHOP were detected by Western blot.Results Compared with group NC and group HC respectively,the cell viability was significantly weakened,the cell apoptosis rates were significantly increased,and the contents of GRP78 and CHOP were significantly increased（P〈0.01）in group NH and group HH.Compared with group NH,the cell viability was significantly weakened,the apoptosis rate was significantly increased,and the contents of GRP78 and CHOP were significantly increased in group HH（P〈0.01）.Conclusion High glucose aggravates neuroblastoma cells hypoxia-reoxygenation injury,which is associated with endoplasmic reticulum chaperones GRP78 and proapoptotic transcription factor CHOP induction.