组织蛋白酶L对脊髓损伤后小胶质细胞极化的影响研究  

Effect of Cathepsin-L on Microglia Polarization After Spinal Cord Injury

作　　者：李名武[1,2] 秦书超 周茹宇 段军 李静静[4] Li Mingwu;Qin Shuchao;Zhou Ruyu;Duan Jun;Li Jingjing(Department of Orthopaedics,Huangshi Central Hospital,Affiliated Hospital of Hubei Polytechnic University,Huangshi 435000,China;Medical College,Wuhan University of Science and Technology,Wuhan 430070,China;Medical College,Hubei Polytechnic University,Huangshi 435000,China;Department of Geriatrics,Huangshi Central Hospital,Affiliated Hospital 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,湖北黄石435000 [2]武汉科技大学医学院,湖北武汉430070 [3]湖北理工学院医学院,湖北黄石435000 [4]黄石市中心医院,湖北理工学院附属医院老年病科,湖北黄石435000

出　　处：《实用骨科杂志》2024年第11期994-1002,共9页Journal of Practical Orthopaedics

基　　金：湖北省科技厅自然科学基金面上项目(2022CFB505);湖北理工学院校级科研项目(22xjz06Y)。

摘　　要：目的从小鼠小胶质细胞在脊髓损伤前后的表型变化入手,寻找通过调控小胶质细胞极化从而影响脊髓功能的基因。方法利用基因表达综合数据库(gene expression omnibus,GEO)的GSE172167数据集,分析小鼠在脊髓损伤前后的脊髓组织单细胞测序数据,采用主成分分析(principal component analysis,PCA)和一致流形近似与投影t-分布随机邻域嵌入(t-distributed stochastic neighbor embedding,t-SNE)降维,获得10个亚群细胞,并筛选出脊髓损伤后小胶质细胞中高表达的差异基因。2023年2月至2023年4月采用BV2细胞和C57BL/6小鼠进行前瞻性研究,验证组织蛋白酶L(cathepsin-L,CTSL)的功能和作用机制。首先将BV2细胞分为7组:对照组,脂多糖(lipopolysaccharide,LPS)组,白细胞介素(interleukin,IL)-4组,阴性对照(NC)-shRNA组,敲低(sh)-CTSL组,NC-过表达(OE)组,OE-CTSL组。动物模型分为5组:假手术(sham)组,脊髓损伤(spinal cord injury,SCI)组,sh-CTSL组,OE-CTSL组,空白组。实时荧光定量聚合酶链反应(quantitative polymerase chain reaction,qPCR)和蛋白质印迹法(western blot,WB)检测CTSL的mRNA和蛋白水平,qPCR检测肿瘤坏死因子(tumour necrosis factor,TNF)-α和IL-1β。流式细胞术检测M1型小胶质细胞(CD86+/CD80+)和M2型小胶质细胞(CD163+/CD206+)占比比例。免疫荧光染色检测小鼠脊髓组织中诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)阳性细胞数和Arg-1阳性细胞数。另外,评估5组小鼠在0、1、2、3、4周的运动神经功能(basso-beattie-bresnahan,BBB)评分。结果体外细胞实验中,对照组M1型小胶质细胞比例、IL-1β、TNF-α和M2型小胶质细胞比例均低于LPS组;LPS组M1型小胶质细胞比例、IL-1β和TNF-α高于IL-4组,而M2型小胶质细胞比例则低于IL-4组;sh-CTSL组M1型小胶质细胞比例、IL-1β和TNF-α均低于LPS组和NC-shRNA组,而M2型小胶质细胞比例则高于这两组;OE-CTSL组M1型小胶质细胞比例、IL-1β和TNF-α均高于IL-4�Objective This study aims to identify genes influencing spinal cord function by regulating microglial cell polarization,based on phenotypic changes in mouse microglia before and after spinal cord injury(SCI).Methods Utilizing the GSE172167 dataset from the gene expression omnibus(GEO),we analyzed single-cell sequencing data of mouse spinal cord tissue pre-and post-SCI.Principal component analysis(PCA)and t-distributed stochastic neighbor embedding(t-SNE)reduced dimensionality,identifying 10 cell subgroups.Subsequently,differential genes highly expressed in microglia post-SCI were isolated.A prospective study from February to April 2023 employed BV2 cells and C57BL/6 mice to validate the function and mechanism of cathepsin-L(CTSL).BV2 cells were categorized into seven groups:control,lipopolysaccharide(LPS),interleukin(IL)-4,negative control(NC)-shRNA,knockdown(Sh)-CTSL,NC-overexpression(OE),and OE-CTSL.The animal model included five groups:sham surgery,SCI,transfection with sh-CTSL adenovirus,transfection with OE-CTSL adenovirus,and a control group.Real-time quantitative polymerase chain reaction(qPCR)and western blotting(WB)assessed CTSL mRNA and protein levels,along with qPCR for tumor necrosis factor-alpha(TNF-α)and IL-1β.Flow cytometry determined M1-type(CD86+/CD80+)and M2-type(CD163+/CD206+)microglia proportions.Immunofluorescence staining quantified iNOS-positive and Arg-1-positive cells in mouse spinal cord tissue.Additionally,the Basso-Beattie-Bresnahan(BBB)scale evaluated motor neuron function in all five mouse groups at 0,1,2,3,and 4 weeks post-injury.Results In vitro,the control group exhibited lower proportions of M1 microglia,IL-1β,TNF-α,and M2 microglia compared to the LPS group.The LPS group showed higher M1 microglia,IL-1β,and TNF-αlevels than the IL-4 group,with a lower M2 microglia proportion.In the sh-CTSL group,M1 microglia,IL-1β,and TNF-αlevels were lower than in the LPS and NC-shRNA groups,while M2 microglia were higher.Conversely,in the OE-CTSL group,M1 microglia,IL-1β,and TNF-αlev

关 键 词：组织蛋白酶L 小胶质细胞极化 炎症反应 单细胞测序 脊髓损伤 

分 类 号：R651.2[医药卫生—外科学]