MicroRNA-181b及Toll样受体4在新生大鼠神经元缺氧缺血损伤中的作用机制研究  

Micro RNA-181b regulates hypoxic-ischemic brain damage through TLR4 signaling pathway

作　　者：阎雯[1] 齐薛浩[2] Wen Yan;Xue-hao Qi(Department of neonatology,Xi’an Children's Hospital,Xi’an,Shanxi 710003,China;Department of NICU,Xi’an Children's Hospital,Xi’an,Shanxi 710003,China)

机构地区：[1]陕西省西安市儿童医院新生儿一科,陕西西安710003 [2]陕西省西安市儿童医院NICU科,陕西西安710003

出　　处：《中国现代医学杂志》2018年第21期21-27,共7页China Journal of Modern Medicine

摘　　要：目的观察Micro RNA-181b(mi RNA-181b)及Toll样受体4(TLR4)对新生大鼠缺氧缺血脑损伤(HIBD)的影响并探讨其潜在机制。方法选取3只1~2日龄新生大鼠脑皮质神经元细胞原代培养7 d,分为对照组和HIBD组。CKK-8法测定原代培养的脑皮质神经元细胞活性,流式细胞术检测细胞凋亡情况,瞬时转染调节细胞内mi RNA-181b及TLR4表达。双荧光素酶实验检测mi RNA-181b对TLR4基因的靶向调控,SYBR Green实时荧光定量PCR观察mi RNA-181b及TLR4 m RNA表达变化。Western blot观察TLR4蛋白在不同组别神经元细胞中的表达。结果与对照组比较,mi RNA-181b在新生大鼠缺氧缺血损伤神经元细胞中表达降低(P<0.05),mi RNA-181b mimic转染后过表达mi RNA-181b能抑制缺血缺氧对新生大鼠神经元细胞的损伤(P<0.05)。双荧光素酶实验证实mi RNA-181b可与TLR4 m RNA 3'-UTR直接结合,发挥对TLR4转录后翻译的抑制作用。进一步机制研究发现,si-TLR4转染后抑制TLR4表达能增加细胞活性,而mi RNA-181b抑制剂anti-mi RNA-181b转染后降低细胞活性,anti-mi RNA-181b与si-TLR4共转染能部分逆转神经元细胞缺血缺氧下的损伤(P<0.05)。结论新生大鼠HIBD神经元细胞中mi RNA-181b能够负性调控TLR4表达发挥一定的神经保护作用。Objective To investigate the effects of MicroRNA-181b(miR-181b)and toll like receptor 4(TLR4)in the progress of Neonatal hypoxic-ischemic brain damage(HIBD)and the underlying mechanism.Methods Brain tissues of 1 or 2 days old SD newborn rats were isolated and cultured as primary neurons.Hypoxia ischemia cell model was established with standardized procedure.Cellular proliferation and cell apoptosis rate of primary cultured cortical neurons were determined by CKK-8 assay and flow cytometry assay,respectively.Expression of miR-181b and TLR4 was modulated by transient transfection,and the activity of TLR4 regulated by miR-181b was evaluated by a dual luciferase reporter assay.Levels of MiR-181b and TLR4 were determined by Real-time quantitative PCR and Western blot.Results Expression of miR-181b was significantly increased in hypoxic-ischemic damaged rat cortical neurons when compared with control group(P<0.05),while overexpression of miR-181b apparently reversed hypoxia-induced cell damage(P<0.05).Dual luciferase reporter assay demonstrated that miR-181b directly targeted the 3’-UTR of the TLR4,resulting in inhibition of the post-transcriptional translation of TLR4.Further mechanism researches revealed that knockdown of TLR4 expression by si-TLR4 transfection significantly upregulated cell viability in rat cortical neurons subjected to hypoxic-ischemic damage,which was abolished by anti-miR-181b transfection treatment.Co-transfection of anti-miR-181b and si-TLR4 in rat cortical neurons were partially attenuated hypoxia induced damage(P<0.05).Conclusions MiR-181b protects rat cortical neurons under hypoxic-ischemic condition through regulating of TLR4.

关 键 词：新生儿缺血缺氧性脑病 MicroRNA-181b TOLL样受体4 

分 类 号：R722.1[医药卫生—儿科]