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

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。

Abstract:

Objective To study the expression and effect of Pim1 in primary cortical neurons after hypoxic-ischemic 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.