微小RNA-211-5p靶向抑制促红细胞生成素肝细胞激酶受体及配体B2信号通路对脊髓神经损伤和功能影响研究  

Functional effects of microRNA-211-5p-targeted inhibition of erythropoietin hepatocyte kinase receptor and ligand B2 signaling pathway on spinal cord nerve injury

作　　者：李名武[1] 钱李慧 孙法瑞[1] 段军[1] 刘亮 Li Mingwu;Qian Lihui;Sun Farui;Duan Jun;Liu Liang(Department of Orthopedic,Huangshi Central Hospital,Affiliated Hospital of Hubei Polytechnic University,Huangshi 435000,China;Medical of Hubei Polytechnic University,Huangshi 435000,China)

机构地区：[1]黄石市中心医院(湖北理工学院附属医院)骨科,黄石435000 [2]湖北理工学院医学院,黄石435000

出　　处：《中国医师进修杂志》2024年第3期212-221,共10页Chinese Journal of Postgraduates of Medicine

基　　金：湖北省自然基金项目(2022CFB505);湖北理工学院校级科研项目(22xjz06Y)。

摘　　要：目的检测微小RNA(miR)-211-5p、促红细胞生成素肝细胞激酶受体B2(EphB2)及促红细胞生成素肝细胞激酶配体B2(ephrin B2)在脊髓损伤(SCI)后脊髓组织以及神经细胞中的表达,探讨其对SCI大鼠神经功能恢复的机制和效果。方法2020年5月至2021年6月采用斯普拉格-道利(SD)大鼠和未受伤的PC12细胞进行前瞻性研究。SD大鼠分为假手术组和SCI组,每组各30只,在术后不同时间点(1、3、7、14、21和28 d)进行Basso-Beattie-Bresnahan(BBB)评分,实时荧光定量聚合酶链反应(qPCR)检测miR-211-5p和Eph/ephrin B2 mRNA的相对表达含量;另将SCI大鼠分为重组慢病毒载体LV-miR-211-5p组(A组)、空慢病毒载体LV-eGFP(B组)、0.9%氯化钠组(C组),每组15只,分别重组慢病毒载体、空慢病毒载体和0.9%氯化钠注射于脊髓损伤处头、尾侧,于术后1、7和14 d收集BBB评分,检测脊髓组织中的miR-211-5p和Eph/ephrin B2 mRNA的相对表达含量,另用免疫荧光染色法检测各组GAP-43和突触素的表达。另外用150μmol/L过氧化氢(H2O2)建立PC12损伤细胞系模型,分别用流式细胞术、Western blot检测不同细胞系的凋亡率和凋亡相关蛋白和含量,双荧光素酶报告基因验证miR-211-5p是否靶向调控EphB2。结果动物实验结果显示,术后不同时间点,SCI组的miR-211-5p在损伤后1、3、7、14、21和28 d水平低于假手术组(0.70±0.03比1.00±0.10、0.60±0.04比1.00±0.05、0.45±0.10比1.00±0.12、0.30±0.06比1.00±0.15、0.20±0.05比1.00±0.13、0.10±0.02比1.00±0.07),EphB2和ephrinB2水平高于假手术组(1.10±0.05比1.00±0.01、1.80±0.01比1.00±0.08、2.30±0.01比1.00±0.10、2.60±0.01比1.00±0.05、2.80±0.01比1.00±0.06、3.00±0.01比1.00±0.07,1.20±0.05比1.00±0.02、1.60±0.01比1.00±0.03、2.10±0.10比1.00±0.01、2.40±0.11比1.00±0.09、2.70±0.13比1.00±0.05、2.90±0.12比1.00±0.03),差异均有统计学意义(P<0.05);术后14 d,A组BBB评分高于B组和C组[(14.0±1.1)分比(8.0±1.1)和(8.2±1.Objective To detect the expression of microRNA(miR)-211-5p,erythropoietin hepatocyte kinase receptor B2(EphB2)and erythropoietin hepatocyte kinase ligand B2(ephrin B2)in spinal cord tissues as well as nerve cells after spinal cord injury(SCI),and to explore their mechanisms and effects on neurological recovery in SCI rats.Methods The study was conducted from May 2020 to June 2021 using Sprague Dawley(SD)rats and PC12 cells.SD rats were divided into sham-operated group and SCI group of 30 rats each,and Basso-Beattie-Bresnahan(BBB)score were performed at different postoperative time points(1,3,7,14,21 and 28 d),and the relative expression of miR-211-5p and Eph/ephrin B2 mRNA was measured by quantitative real-time polymerase chain reaction(qPCR);the SCI rats were divided into recombinant lentiviral vector LV-miR-211-5p group(group A),empty lentiviral vector LV-eGFP(group B)and saline group(group C),with 15 rats in each group,respectively.The recombinant lentiviral vector,empty lentiviral vector and saline were injected on the cephalic and caudal sides of the spinal cord injury,and the relative expression of miR-211-5p and Eph/ephrin B2 mRNA in the spinal cord tissue was measured at 1,7 and 14 d after surgery.In addition,a PC12 injury cell line model was established with 150μmol/L hydrogen peroxide(H2O2),and the apoptosis rate and apoptosis-related proteins and contents of different cell lines were detected by flow cytometry and Western blot,respectively.MiR-211-5p was verified to target EphB2 by dual luciferase reporter gene.Results The results of the animal experiments showed that at different postoperative time points,the miR-211-5p levels in the SCI group were lower than those in the SHAM group:0.70±0.03 vs.1.00±0.10,0.60±0.04 vs.1.00±0.05,0.45±0.10 vs.1.00±0.12,0.30±0.06 vs.1.00±0.15,0.20±0.05 vs.1.00±0.13,0.10±0.02 vs.1.00±0.07.In contrast,levels of Eph/ephrin B2 were higher in the SCI group compared to the SHAM group:1.10±0.05 vs.1.00±0.01,1.80±0.01 vs.1.00±0.08,2.30±0.01 vs.1.00±0.10,2.60±

关 键 词：脊髓损伤 微RNAS 促红细胞生成素肝细胞激酶 功能 

分 类 号：R651.2[医药卫生—外科学]