中文摘要：

目的 探讨miR-181b在氧糖剥夺（OGD）致N2As神经瘤细胞缺血损伤中的作用，及其对热休克蛋白（HSP）A5表达的调节。方法 应用N2A细胞OGD模型模拟神经细胞缺血损伤，MTT比色法检测N2A细胞生存率，免疫印迹法检测HSPA5蛋白表达水平，实时定量PCR法检测miR-181b和HSPA5 mRNA表达水平，荧光素酶报告基因技术检测miR-181b对HSPA5 mRNA的直接调控作用。 结果 miR-181b在OGD致N2A细胞缺血损伤中表达水平明显降低（n=5）；在OGD致N2A细胞缺血损伤过程中，通过上调或抑制miR-181b的表达水平可以显著影响N2A细胞的生存率（n=6）；而在非OGD条件下，miR-181b表达水平的改变对N2A细胞活力无影响；miR-181b表达水平的改变可显著影响HSPA5蛋白表达水平（n=3），而非HSPA5的mRNA水平；共转染miR181b前体（pre-miR-181b）或miR-181b抑制剂（anti-miR-181b）可显著抑制或增高含有HSPA5 mRNA 3’-UTR的荧光素酶报告基因的活性（n=5）。 结论 miR-181b通过负性调节HSPA5的蛋白表达水平，在OGD致N2A神经细胞缺血性损伤中发挥重要作用。

英文摘要：

Objective To explore the role of microRNA-181b（miR-181b ）in oxygen-glucose deprivation （OGD）-induced N2A cell ischemic injury and its regulation on HSPA5 protein levels. Methods Using N2A cell OGD model to mimic ischemic injuryin vitro, the extent of N2A cell survival rate was assessed by thiazolyl blue tetrazolium bromide （MTT） assay. The heat shock protein A5 （HSPA5 ） levels and the expression levels of miR-181b and HSPA5 mRNA were determined by using Western blotting and Real-time PCR, respectively. Luciferase reporter assay was performed to identify the direct binding of miR-181b with 3’-UTR of HSPA5 mRNA. Results The miR-181b expression level decreased significantly （P〈0.05, n=5 per group） in OGD-treated N2A cells. Under the condition of OGD but not in non-OGD condition, alteration of miR-181b expression level by transfection with pre-miR-181b or anti-miR-181b significantly affected N2A cell survival rate（-n=6）. Accordingly, the changes of miR-181b levels significantly altered HSPA5 protein levels（n=3）, but not the expression levels of HSPA5 mRNA. In addition, the results of luciferase reporter assay indicated that co-transfection of the luciferase reporters with pre-miR-181b or anti-miR-181b resulted in the inhibition or enhancement of the luciferase activities of luciferase expressing plasmid containing 3’-UTR of HSPA5 mRNA（n=5）. Conclusions miR-181b plays an important role in N2A cell ischemic injury through negatively regulating HSPA5 protein level, which may provide a potential therapeutic target for ischemic stroke in miRNA levels.