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目的研究内质网应激(ERS)GRP78-PERK-CHOP通路分子在不同高氧暴露时间下参与高氧所致新生鼠脑白质凋亡的情况。方法新生6日龄C57BL/6小鼠共14窝,每窝8~10只,随机分为高氧组(80%O2)及对照组(21%O2)各7窝。分别于高氧6、12、24、48 h以及高氧48 h后4、7、22天(P12、P15、P30)断头取脑,并与对照组比较。采用RT-PCR和蛋白质印迹法检测高氧后GRP78、PERK、CHOP mRNA和蛋白表达变化,TUNEL法检测小鼠脑白质细胞凋亡情况,透射电镜观察小鼠脑白质髓鞘化改变。结果与对照组相比,高氧6 h后,GRP78 mRNA和蛋白表达增高(P=0.001,0.004),12、24、48 h差异均无统计学意义(P>0.05)。高氧6 h后,PERK mRNA和蛋白表达高于对照组(P=0.001,0.001),余时间点组间比较,差异均无统计学意义(P>0.05)。高氧12 h后,CHOP mRNA和蛋白表达高于对照组(P=0.011,0.003),余时间点组间比较差异均无统计学意义(P>0.05)。与对照组比较,P15高氧组未见成熟的髓鞘结构,P30高氧组髓鞘平均厚度和轴突直径明显降低,出现异常的未折叠的髓鞘和异常的环形结构。P12、P15高氧组凋亡指数明显高于对照组(P=0.001,0.030)。结论高氧暴露可导致新生小鼠脑白质损伤,GRP78-PERK-CHOP通路启动的凋亡途径可能参与其中。
Objective To investigate the role of GRP78-PERK-CHOP pathway in endoplasmic reticulum stress (ERS) in the apoptosis of neonatal white matter induced by hyperoxia in different periods of hyperoxia exposure. Methods Newborn 6-day-old C57BL / 6 mice were divided into 8 groups (n = 10 in each group), and were randomly divided into 7 groups (80% O2) and control group (21% O2) The brain was decapitated at 6, 12, 24, and 48 h after hyperoxia and at 48 and 48 h after hypoxia, respectively, and compared with the control group. The mRNA and protein expressions of GRP78, PERK and CHOP were detected by RT-PCR and Western blotting. The apoptosis of mouse white matter cells was detected by TUNEL method. The changes of white matter myelination were observed by transmission electron microscopy. Results Compared with the control group, the expression of GRP78 mRNA and protein increased after 6 h of hyperoxia (P = 0.001 and 0.004), but no significant difference at 12, 24 and 48 h (P> 0.05). After 6 h of hyperoxia, the expression of PERK mRNA and protein were higher than those of the control group (P = 0.001 and 0.001). There were no significant differences in the PERK mRNA and protein expression between the two groups at any time point (P> 0.05). After 12 h of hyperoxia, the expression of CHOP mRNA and protein were higher than that of the control group (P = 0.011,0.003), but there was no significant difference between the two groups (P> 0.05). Compared with the control group, there was no mature myelin structure in P15 hyperoxia group. The average thickness of myelin sheath and axon diameter in P30 hyperoxia group were significantly decreased, with abnormal unfolded myelin and abnormal ring structure. The apoptotic index in P12 and P15 hyperoxia group was significantly higher than that in control group (P = 0.001,0.030). Conclusion Hyperoxia exposure can lead to brain white matter injury in neonatal mice, and the apoptotic pathway of GRP78-PERK-CHOP pathway may be involved.