摘要
目的:探讨miR-199a对缺氧/复氧诱导的大鼠脑皮层神经元细胞活力及凋亡的影响。方法:在体外培养大鼠脑皮层神经元细胞,采用缺氧/复氧的方式处理细胞建立细胞损伤模型并记作Model组;将正常培养的细胞作为对照(Con)组。分别将空载体、miR-199a mimic转染至大鼠脑皮层神经元细胞,建立缺氧/复氧细胞损伤模型,分别记作Model+NC组或Model+miR-199a mimic组。将miR199a mimic转染至大鼠脑皮层神经元细胞后加入PI3K-AKT信号通路抑制剂LY294002处理,随后建立缺氧/复氧细胞损伤模型,记作Model+miR-199a mimic+LY294002组。采用反转录PCR法检测miR-199a的表达量,应用流式细胞仪检测细胞周期及细胞凋亡率,采用MTT法检测细胞活力,采用蛋白质印迹法检测cleaved-caspase3、pro-caspase3、p-PI3K、p-AKT蛋白表达量。结果:与Con组比较,Model组miR-199a的表达水平、细胞活力、S期细胞比例及pro-caspase3、p-PI3K、p-AKT蛋白表达水平均显著降低,G_(0)~G_(1)期细胞比例、细胞凋亡率及cleaved-caspase3蛋白表达水平均显著提高,差异均有统计学意义(均P<0.001)。与Model+NC组比较,Model+miR-199a mimic组细胞活力、S期细胞比例及pro-caspase3、p-PI3K、p-AKT蛋白表达水平均显著提高,G_(0)~G_(1)期细胞比例、细胞凋亡率及cleaved-caspase3蛋白表达水平均显著降低,差异均有统计学意义(均P<0.001)。与Model+miR-199a mimic组比较,Model+miR-199a mimic+LY294002组细胞活力、S期细胞比例及procaspase3蛋白表达水平均显著降低,G_(0)~G_(1)期细胞比例、细胞凋亡率及cleaved-caspase3蛋白表达水平均显著提高,差异均有统计学意义(P<0.05)。结论:MiR-199a过表达可改善缺氧/复氧诱导的大鼠脑皮层神经元细胞活力并抑制细胞凋亡,从而减轻细胞损伤;这些生物学效应可能是通过激活PI3K-AKT信号通路产生的。
Objective:To explore the effect of miR-199a on the viability and apoptosis of rat cerebral cortical neurons cells induced by hypoxia/reoxygenation.Methods:Rat cerebral cortical neurons cells were cultured in vitro and treated with hypoxia/reoxygenation to establish the cell injury model and recorded as Model group,and normal cultured cells were used as control(Con)group.Empty vector and miR-199a mimic were respectively transfected into rat cerebral cortical neurons cells,and then hypoxia/reoxygenation cell injury models were established,which were recorded as Model+NC group or Model+miR-199a mimic group,respectively.After miR-199a mimic was transfected into rat cerebral cortical neurons cells,PI3K-AKT signaling pathway inhibitor LY294002 was added for treatment,and then hypoxia/reoxygenation cell injury model was established and recorded as Model+miR-199a mimic+LY294002 group.Then,the expression of miR-199a was detected by RT-PCR,the cell cycle and apoptosis rate were detected by flow cytometry,the cell viability was detected by MTT,and the expression of Cleaved-caspase3,pro-caspase3,p-PI3K and p-AKT proteins were detected by Western blot.Results:Compared with Con group,the expression level of miR-199a,cell viability,S-phase cell proportion and the expression levels of pro-caspase3,p-PI3K and p-AKT protein in Model group were significantly lower,while the cell proportion of G_(0)-G_(1) phase,the apoptosis rate and the expression level of cleaved-caspase3 protein were significantly higher in Model group(P<0.05).Compared with the Model+NC group,the cell viability,the proportion of S-phase cells and the expression levels of pro-caspase3,p-PI3K and p-AKT protein in the Model+miR-199a mimic group were significantly increased,while the proportion of G_(0)-G_(1) phase cells,the rate of apoptosis and the expression level of cleaved-caspase3 protein were significantly decreased(P<0.05).Compared with the Model+miR-199a mimic group,the cell viability,the proportion of S-phase cells and the expression level of pro-caspase3 protein in the Model+miR-199a mimic+LY294002 group were significantly decreased,and the proportion of G_(0)-G_(1) phase cells,the apoptosis rate and the expression level of cleaved-caspase3 protein were significantly increased(P<0.05).Conclusion:The overexpression of miR-199a could improve the viability of rat cerebral cortical neurons cells induced by hypoxia/reoxygenation and inhibit cell apoptosis,thereby reducing cell damage.The above biological effects may be achieved by activating PI3K-Akt signaling pathway.
作者
巩雨
樊嘉欣
高震
王虎清
吴海琴
GONG Yu;FAN Jiaxin;GAO Zhen;WANG Huqing;WU Haiqin(Department of Neurology,Second Affiliated Hospital,Xi’an Jiaotong University,Xi’an 710004,China)
出处
《临床与病理杂志》
CAS
2022年第5期1028-1035,共8页
Journal of Clinical and Pathological Research
基金
陕西省重点研发计划(2017SF-181)。
