miR-381-3p通过靶向调控TP53表达对大鼠脊髓损伤后修复的作用及其机制研究  

The effect and mechanism of miR-381-3p on the repair of spinal cord injury in rats via the regulation of TP53

作　　者：白夜 王勇 张文进 许战武 陈庆贺 Bai Ye;Wang Yong;Zhang Wenjin(Department of Orthopedics,962 Hospital of Joint Logistic Support Force,Harbin 150000)

机构地区：[1]黑龙江省哈尔滨市联勤保障部队第九六二医院骨科

出　　处：《卒中与神经疾病》2023年第5期461-473,共13页Stroke and Nervous Diseases

摘　　要：目的探讨微小RNA-381-3p(MicroRNA-381-3p,miR-381-3p)对脊髓损伤(Spinal cord injury,SCI)大鼠继发性损伤的修复机制以及其对肿瘤抑制蛋白基因(Tumor protein p53,TP53)表达水平的调控机制。方法双荧光素酶以及蛋白质免疫共沉淀检测miR-381-3p和TP53的作用;用改良Allen′s重物垂直撞击法复制大鼠SCI模型;实验动物分为假手术(Sham)组、SCI组、miR-381-3p-mimic(mimic)组、mimic+pc DNA3.1 TP53(mimic+pc)组,每组各10只;mimic组大鼠尾静脉注射miR-318-3p mimic;mimic+pc组大鼠尾静脉注射miR-318-3p mimic和pc DNA3.1 TP53;Sham组和SCI组大鼠尾静脉注射miR-318-3p mimic-NC和pc DNA3.1 TP53-NC;检测各组大鼠的运动功能评分(Basso beattie bresnahan,BBB)评分;脱氧核糖核苷酸末端转移酶介导的缺口末端标记试剂盒(Terminal-deoxynucleoitidyl transferase-mediated nick end labeling,TUNEL)检测各组大鼠脊髓组织的细胞凋亡;超氧化物阴离子二氢乙啶(Dihydroethidium,DHE)荧光探针检测脊髓组织中活性氧(Reactive oxygen species,ROS)自由基的水平;免疫荧光检测各组大鼠脊髓组织中小胶质细胞的表型转换;酶联免疫吸附(Enzyme-linked immunosorbent assay,ELISA)检测各组大鼠血清中超氧化物歧化酶(Superoxide dismutase,SOD)、丙二醛(Malondialdehyde,MDA)、白细胞介素-1β(Interleukin-1β,IL-1β),IL-6,IL-4和IL-10的水平;彗星实验检测各组脊髓组织中的DNA损伤;Western blot实验检测各组大鼠脊髓组织中肿瘤抑制蛋白基因(Tumor protein p53,TP53)、核转录因子(Nuclear factorκB,NF-κB)、磷酸化组蛋白(γ-Histone family 2A variant,γ-H2AX)、B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl2-associated x protein,Bax)水平。结果双荧光素酶以及蛋白质免疫共沉淀显示miR-381-3p调控TP53的表达水平。与Sham组比较,SCI组、mimic组、mimic+pc组大鼠的BBB评分、血清中SOD的活性、脊髓组织中M2型细胞的比例、Bcl-2的表达水平明显降低,脊髓组织中的细ObjectiveTo investigate the repair mechanism of microRNA-381-3p(miR-381-3p)on secondary injury in spinal cord injury(SCI)rats and its regulatory mechanism on the expression of tumor protein p53(TP53).Methods Double luciferase and protein immunoprecipitation were used to detect the effect of miR-381-3p and TP53.The modified Allen's weight vertical impact method was used to establish the rat SCI model.The experimental animals were divided into the sham operation group(Sham),SCI group,miR-381-3p-mimic(mimic)group,and mimic plus pc DNA3.1 TP53(mimic+pc)group(n=10).Rats in the mimic group were injected with miR-318-3p mimic through the tail vein.Rats in the mimic+pc group were injected with miR-318-3p mimic and pc DNA3.1 TP53.Rats in the Sham group and SCI group were injected with miR-318-3p mimic-NC and pc DNA3.1 TP53-NC via the tail vein.The basso beattie bresnahan(BBB)score of rats in each group was detected.Terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL)was used to detect apoptosis in spinal cord tissue.Dihydroethidium(DHE)was used to detect the content of reactive oxygen species(ROS).Immunofluorescence was used to detect the morphological transformation of microglia in spinal cord tissue.Enzyme-linked immunosorbent assay(ELISA)was used to detect the contents of superoxide dismutase(SOD),malondialdehyde(MDA),interleukin-1β(IL-1β),IL-6,IL-4 and IL-10 in the serum of rats in each group.A comet assay was used to detect DNA injury in spinal cord tissue.Western blotting was used to detect the expression of TP53,NF-κB,γ-histone family 2A variant(γ-H2AX),B lymphocyte tumor-2(Bcl-2)and Bcl-2-associated X protein(Bax)in spinal cord tissue.Results The results of double luciferase and protein immunoprecipitation assays showed that miR-381-3p regulated the expression of TP53.The BBB score,SOD activity in serum,proportion of M2 cells in spinal cord tissue,and expression of Bcl-2 in spinal cord tissue were significantly decreased in the SCI group,mimic group and mimic+pc group compared with the sham

关 键 词：微小RNA-381-3p 肿瘤抑制蛋白基因 脊髓损伤 氧化和炎症应激 DNA损伤 

分 类 号：R651.2[医药卫生—外科学]