视神经脊髓炎谱系疾病患者外周血TIMP1和MMP9及miR-363-5p的表达变化及其与血脑屏障破坏的关系  

The expression levels of peripheral blood matrix metalloproteinase 9,inhibitor of matrix metallo proteinases 1,and miR-363-5p genes,and their associations with blood brain barrier dysfunction in neuromyelitis optica spectrum disorders

作　　者：闫红静 李彦梅[1] 申晓玲[1] 马丽芬 王敏 习旭涛 杜娟 陈卫峰 李彬[3] YAN Hongjing;LI Yanmei;SHEN Xiaoling;MA Lifen;WNAG Min;XI Xutao;DU Juan;CHEN Weifeng;LI Bin(不详;Department of Neurology,The Second Hospital of Hebei Medical University,Shijiazhuang 056001,China)

机构地区：[1]邯郸市第一医院神经内二科,056000 [2]邯郸市中心医院神经外科,056000 [3]河北医科大学第二医院神经内科,056001

出　　处：《中国神经免疫学和神经病学杂志》2023年第5期309-315,共7页Chinese Journal of Neuroimmunology and Neurology

基　　金：河北省医学科学研究课题计划(项目编号:20210117)。

摘　　要：目的探讨视神经脊髓炎谱系疾病(NMOSD)患者外周血金属蛋白酶抑制剂1(TIMP1)mRNA及血浆TIMP1蛋白、基质金属蛋白酶9(MMP9)及miR-363-5p的表达变化及其与血脑屏障(BBB)破坏的关系。方法选取2019年9月至2021年11月河北医科大学第二医院收治的NMOSD患者30例,另选择健康体检者作为健康对照组10例。采用ELISA方法检测血浆TIMP1、MMP9的水平。采用qPCR法检测外周血单个核细胞(PBMC)TIMP1 mRNA和血浆miR-363-5p水平。根据脑脊液/血白蛋白商(Qalb)将NMOSD患者分为BBB正常组与BBB破坏组,分别比较NMOSD组与正常对照组以及BBB正常组与BBB破坏组TIMP1、MMP9及miR-363-5p表达差异,采用相关性分析评估其与BBB破坏的关系。采用双萤光素酶报告基因检测技术验证miR-363-5p与TIMP1靶向结合关系。通过慢病毒载体感染JURKA T细胞并分为4组:未转染miR-363-5p模拟物的正常对照组,转染miR-363-5p模拟物组,未转染miR-363-5p抑制剂的正常对照组,转染miR-363-5p抑制剂组。采用qPCR检测JURKA T细胞TIMP1 mRNA及MMP9 mRNA的表达,采用Western-blot技术检测JURKA T细胞TIMP1、MMP9蛋白表达的水平。结果(1)NMOSD组患者PBMC TIMP1 mRNA、血浆miR-363-5p、血浆TIMP1和MMP9蛋白水平高于健康对照组(P<0.01或P<0.05)。NMOSD组和健康对照组MMP9/TIMP1比值比较差异无统计学意义(P>0.05)。(2)NMOSD患者BBB破坏组MMP9/TIMP1比值高于BBB正常组(P<0.05)。NMOSD患者MMP9/TIMP1比值与Qalb呈中等正相关(r=0.505,P<0.01)。NMOSD患者血浆miR-363-5p与PBMC TIMP1 mRNA相对表达水平无相关性(r=-0.33,P>0.05),与血浆TIMP1蛋白表达水平呈中等负相关(r=-0.5157,P<0.01)。miR-363-5p可与TIMP13'UTR区结合,在JURKA T细胞中,转染miR-363-5p模拟物后TIMP1 mRNA和蛋白质表达水平下降(P<0.01,P<0.05)。结论NMOSD患者PBMC TIMP1 mRNA以及血浆TIMP1蛋白、MMP9蛋白、miR-363-5p表达升高;miR-363-5p可负调控JURKA T细胞TIMP1的表达;血浆MMP9/TIMP1比值升高可能在NMOSD患�Objective To explore the expression levels of peripheral blood metalloproteinase inhibitor 1(TIMP1)mRNA and TIMP1 protein,plasma matrix metalloproteinase 9(MMP9),and plasma miR-363-5p and their relationship with blood-brain barrier(BBB)dysfunction in neuromyelitis optica spectrum disorders(NMOSD).Methods Thirty NMOSD patients were recruited from September 2019 to November 2021 in the Second Hospital of Hebei Medical University,and 10 healthy controls were selected as the healthy control group.Plasma TIMP1 and MMP 9 were detected using enzyme linked immunosorbent assay(ELISA).Peripheral blood mononuclear cell(PBMC)TIMP1 mRNA and plasma miR-363-5p levels were measured by quantitative real-time PCR(qPCR).Patients were divided into the BBB normal group and BBB destruction group,based on albumin quotient(Qalb)status.Levels of TIMP 1,MMP 9,and miR-363-5p were compared between the NMOSD and control group,and between the BBB normal and BBB destruction group.Correlation analysis was used to assess their relationships with BBB destruction.Dual-luciferase reporter gene assay was used to verify the binding relationship between miR-363-5p and TIMP 1 targeting.Lentivirus titration was performed on the Jurkat T cells line.The cells were divided into 4 groups:normal control group not transfected with miR-363-5p mimic,transfected with miR-363-5p mimic,normal control group not transfected with miR-363-5p inhibitor,and transfected with miR-363-5p inhibitor.The RT-qPCR was performed to determine the mRNA expression levels of MMP9 and TIMP1.Western-blot analysis was used to detect protein levels of MMP9,and TIMP1.Results(1)NMOSD patients PBMC TIMP1 mRNA,plasma miR-363-5p,plasma TIMP 1 and MMP9 protein levels were higher than those in healthy controls(P<0.01 or P<0.05).The difference in the MMP9/TIMP1 ratio between the NMOSD patient group and control group did not reach statistical significance(P>0.05).(2)The expression of MMP9/TIMP1 ratio was higher in the BBB disrupted group than that in the BBB normal group(P<0.05).The MMP9/TIMP1 r

关 键 词：视神经脊髓炎谱系疾病 微RNAs miR-363-5p 金属蛋白酶1组织抑制剂 基质金属蛋白酶9 血脑屏障 

分 类 号：R744.5[医药卫生—神经病学与精神病学]