Pim1在体外培养大脑皮层神经元氧糖剥夺/复氧损伤中的表达及作用  

Expression and role of Pim1 in cultured cortical neurons with oxygen-glucose deprivation/reoxygen injury

作　　者：刘俊燕 王克煊 黄凌依 万彬 赵国英 赵凤艳 LIU Jun-Yan;WANG Ke-Xuan;HUANG Ling-Yi;WAN Bin;ZHAO Guo-Ying;ZHAO Feng-Yan(Department of Neonatology,Binzhou Medical University Hospital,Binzhou,Shandong 256600,China)

机构地区：[1]滨洲医学院附属医院新生儿科,山东滨州256600 [2]四川大学口腔医学院,四川成都610041 [3]四川大学华西第二医院儿科/出生缺陷与相关妇儿疾病教育部重点实验室,四川成都610041

出　　处：《中国当代儿科杂志》2020年第5期512-518,共7页Chinese Journal of Contemporary Pediatrics

基　　金：国家自然科学基金(81971429);四川大学大学生创新训练计划基金(C2019104355)。

摘　　要：目的探讨原代培养大脑皮层神经元缺氧缺血损伤后Pim1的表达及其对细胞凋亡的作用。方法取新生1日龄C57BL/6小鼠的大脑皮层神经元进行原代培养,培养第8天更换无糖无血清DMEM培养基,于1%O2条件下培养3 h后,换为正常条件下培养,即氧糖剥夺/复氧(OGD/R)处理,以模拟体内神经元缺氧缺血状态。分别于OGD/R后0、6、12、24 h收集细胞,同时设立神经元正常培养组。原代培养神经元中分别转染Pim1过表达质粒或空质粒,然后分别进行正常培养或OGD/R处理,分别命名为Pim1组、对照组、OGD/R组和OGD/R+Pim1组;采用Real-time PCR检测Pim1 mRNA的表达,采用Western blot检测Pim1蛋白和凋亡相关蛋白活化的半胱氨酸蛋白酶(CC3)的表达。采用TUNEL法检测细胞凋亡。结果Real-time PCR和Western blot结果显示,与正常组相比,OGD/R后神经元中Pim1 mRNA及其蛋白表达均显著降低,且均从OGD/R后0 h开始下降,12 h最低,24 h回升但仍维持在较低水平(P<0.05)。Pim1过表达转染使神经元中Pim1蛋白水平增加。Pim1+OGD/R组CC3表达水平明显低于OGD/R组,Pim1+OGD/R组细胞凋亡率也较OGD/R组显著减少(P<0.01)。结论缺氧缺血损伤引起体外培养神经元中Pim1表达下降。过表达的Pim1可以抑制OGD/R诱导的神经元凋亡。Objective To study the expression and effect of Pim1 in primary cortical neurons after hypoxicischemic injury.Methods Cortical neurons were isolated from 1-day-old C57BL/6 mice and cultured in neurobasal medium.On the 8th day of neuron culture,cells were subjected to oxygen-glucose deprivation/reoxygen(OGD/R)treatment to mimic in vivo hypoxic injury of neurons.Briefly,medium were changed to DMEM medium,and cells were cultured in 1%O2 for 3 hours and then changed back to normal medium and conditions.Cells were collected at 0 hour,6 hours,12 hours and 24 hours after OGD/R.Primary neurons were transfected with Pim1 overexpression plasmid or mock plasmid,and then were exposed to normal conditions or OGD/R treatment.They were named as Pim1 group,control group,OGD/R group and OGD/R+Pim1 group respectively.Real-time PCR was used to detect Pim1 mRNA expression.Western blot was used to detect the protein expression of Pim1 and apoptotic related protein cleaved caspase 3(CC3).TUNEL staining was used to detect cell apoptosis.Results Real-time PCR and Western blot results showed that Pim1 mRNA and protein were significantly decreased in neurons after OGD/R.They began to decrease at 0 hour after OGD/R,reached to the lowest at 12 hours after OGD/R,and remained at a lower level at 24 hours after OGD/R(P<0.01).Overexpression of Pim1 significantly upregulated the protein level of Pim1.Under OGD/R conditions,the CC3 expression and the apoptosis rate in cells of the Pim1 group were significantly lower than in un-transfected cells(P<0.01).Conclusions Hypoxic-ischemic injury may decrease Pim1 expression in neurons.Overexpressed Pim1 may inhibit apoptosis induced by OGD/R.

关 键 词：脑损伤 细胞凋亡 缺氧缺血 Pim1 神经元 

分 类 号：R742[医药卫生—神经病学与精神病学]