摘要
目的在体外培养的SH-SY5Y细胞和IMR-32细胞中,观察microRNA-25(miR-25)对缺血/再灌注(I/R)损伤诱导的细胞凋亡的作用及其可能机制。方法在体外培养的人SH-SY5Y细胞和IMR-32细胞中,用氧葡萄糖剥夺/再恢复(OGDR)模拟脑缺血/再灌注损伤。合成miR-25过表达片段,并构建慢病毒载体质粒,用脂质体将载体质粒转导入细胞中。MTT法检测细胞活力、TUNEL法检测凋亡、RT-PCR、Real-time PCR和Western blotting分别检测目的基因mRNA和蛋白的表达情况。结果与正常培养组相比,OGDR组中细胞内miR-25表达下调,细胞活力明显下降,凋亡明显增多,同时Bax、Caspase-3 mRNA和蛋白表达上调,Bcl-2 mRNA和蛋白表达下调(n=3,P <0. 05);而与OGDR组相比,过表达miR-25组内细胞活力明显升高,凋亡明显减少,同时Bax、Caspase-3mRNA和蛋白表达下降Bcl-2 mRNA和蛋白表达升高(n=3,P <0. 05)。结论 I/R损伤后miR-25表达上调可能通过Bax/Bcl-2-Caspase-3途径进而抑制I/R损伤诱导的凋亡,为I/R损伤的临床治疗提供潜在的治疗靶点。
Objective To evaluate the role and the possible mechanisms of microRNA-25（ miR-25） in regulating oxygen-glucose deprivation（ OGD）/reperfusion（ OGDR）-induced cell apoptosis in cultured SH-SY5 Y and IMR-32 cell.Methods The OGD model was constructed in human SH-SY5 Y and IMR-32 cells to mimic I/R injury. Fragments mediated miR-25 overexpression,mi-R25 mimics,were synthesized and constructed into recombined lentivirus plasmid.Using lipofectamine,the plasmids were transfected into the cells. The cell viability and apoptosis were detected with MTT assay and TUNEL. The mRNA and protein expression of the target genes were detected using RT-PCR,Real-time PCR and Western blotting respectively. Results Compared with the normal group,miR-25 was down-regulated in OGDR model group with decreased cell viability and increased apoptosis. Meanwhile,mRNA and protein expression of Bax and Caspase-3 were increased,but with decreased expression of Bcl-2（ n = 3,P〈0. 05）. Compared with the OGDR group,cell viability was increased and the apoptosis was decreased in miR-25 transfection group,with decreased expression of Bax and Caspase-3,as well as increased expression of Bcl-2（ n = 3,P〈0. 05）. Conclusion Taken together,our result indicate that up-regulation of miR-25 inhibits cerebral I/R injury-induced apoptosis through regulating Bax/Bcl-2 and Caspase-3 signaling pathway,which will provide a promising therapeutic target for cerebral ischemia/reperfusion injury.
作者
张军峰
赵朝华
郝佳晖
房智超
徐仓宝
徐曦
ZHANG Jun-feng;ZHAO Zhao-hua;HAO Jia-hui;FANG Zhi-chao;XU Cang-bao;XU Xi(Shanxi Key Laboratory of lschemic Cardiovascular Disease,Xi＇an Medical University,Xi＇an 710021,China;Department of Human Anatomy,Xi＇an Medical University,Xi＇an 710021,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2018年第5期584-590,共7页
Acta Anatomica Sinica
基金
陕西省科技厅自然科学基础研究计划(2018JM-7066)
陕西省缺血性心血管疾病重点实验室开放基金(2017ZDKF01)
陕西省大学生创新训练项目(G201711840002
G201711840006)
西安医学院国科金培育基金(2017GJFY25)
西安医学院配套基金(2016PT06)
陕西省优势学科建设经费(陕教位[2014]3号-1001)
