目的:探讨激素性股骨头坏死(steroid-induced osteonecrosis of the femoral head,SONFH)滑膜病变的分子机制.方法:①转录组测序及生物信息学分析.收集9例接受全髋关节置换术的患者(SONFH患者3例、髋骨关节炎患者3例和股骨颈骨折患者3例)术中切除的髋关节滑膜组织,进行转录组测序和生物信息学分析,筛选SONFH滑膜病变核心基因.②滑膜组织检测.根据标本来源将髋关节滑膜组织分为SONFH组、髋骨关节炎组和股骨颈骨折组.观察各组髋关节滑膜组织的组织形态,采用实时定量PCR检测3组髋关节滑膜组织中SONFH滑膜病变核心基因的mRNA相对表达量,采用蛋白质印迹法和免疫组织化学染色法检测3组髋关节滑膜组织中SONFH滑膜病变核心基因的蛋白相对表达量.采用免疫荧光染色法检测SONFH滑膜病变的靶细胞.③细胞验证.培养大鼠滑膜成纤维细胞,构建滑膜炎细胞模型.采用实时定量PCR及蛋白质印迹法检测空白细胞(空白细胞组)和滑膜炎模型细胞(模型细胞组)中SONFH滑膜病变核心基因的表达.结果:①转录组测序和生物信息学分析结果.经对不同患者来源的髋关节滑膜组织进行差异基因分析,共筛选出1001个与SONFH髋关节滑膜病变相关的基因,这些基因与免疫反应和外泌体有关,其中干扰素调节因子(interferon regulatory factor,IRF)4和IRF7是SONFH髋关节滑膜病变的核心基因,两者均为参与Ⅰ型干扰素应答的关键转录因子.②滑膜组织形态观察结果.苏木素-伊红染色显示,股骨颈骨折组髋关节滑膜组织形态正常,无细胞增生、肥大或间质水肿;髋骨关节炎组髋关节滑膜组织细胞增生,有少量新生血管和细胞聚集;SONFH组髋关节滑膜组织细胞大量增殖和聚集,有新生血管.③滑膜组织中SONFH滑膜病变核心基因表达检测结果.SONFH组髋关节滑膜组织中IRF4、IRF7、干扰素-α(interferon-α,IFN-α)的mRNA相对表达量均高于股骨颈骨折组和髋骨关节炎组(P=0.000,P=0.000,P=0.000;P=0.001,P=0.000,P=0.036),髋骨关节炎组髋关节滑膜组织中IRF4、IRF7、IFN-α的mRNA相对表达量均高于股骨颈骨折组(P=0.000,P=0.000,P=0.000).IRF4、IRF7、IFN-α蛋白在SONFH组髋关节滑膜组织中高表达,SONFH组髋关节滑膜组织中IRF4、IRF7、IFN-α的蛋白相对表达量均高于股骨颈骨折组和髋骨关节炎组(P=0.001,P=0.000,P=0.000;P=0.000,P=0.014,P=0.000),髋骨关节炎组髋关节滑膜组织中IRF4、IRF7、IFN-α的蛋白相对表达量均高于股骨颈骨折组(P=0.002,P=0.005,P=0.000).④SONFH滑膜病变靶细胞检测结果.SONFH组髋关节滑膜组织中IRF7、钙黏附蛋白11(cadherin-11,CDH-11)的蛋白相对表达量均高于股骨颈骨折组和髋骨关节炎组(P=0.001,P=0.000;P=0.000,P=0.001),髋骨关节炎组髋关节滑膜组织中IRF7、CDH-11的蛋白相对表达量高于股骨颈骨折组(P=0.001,P=0.000).滑膜成纤维细胞为SONFH滑膜病变的靶细胞.⑤细胞验证结果.模型细胞组IRF4、IRF7、IFN-α的mRNA和蛋白相对表达量均高于空白细胞组(P=0.001,P=0.002,P=0.000;P=0.001,P=0.007,P=0.000).结论:SONFH滑膜病变与免疫炎症反应关系密切,滑膜成纤维细胞可能是SONFH滑膜病变的靶细胞,IRF4和IRF7可能是其潜在的靶点.
Molecular mechanisms of synovial lesions in steroid-induced osteonecrosis of the femoral head:an experimental study
Objective:To investigate the molecular mechanisms of synovial lesions in steroid-induced osteonecrosis of the femoral head(SONFH).Methods:①RNA sequencing(RNA-seq)and bioinformatics analysis.The hip synovial tissues were collected from patients with SONFH(3 ones),hip osteoarthritis(HOA)(3 ones)and femoral neck fracture(FNF)(3 ones),respectively,during the total hip arthroplas-ty for RNA-seq and bioinformatics analysis to screen the core genes of synovial lesions in SONFH.②Synovial tissues detection.The hip syn-ovial tissue specimens were divided into SONFH group,HOA group,and FNF group based on their sources,and then were sectioned and stained with hematoxylin-eosin(HE)for observing the morphology.Furthermore,the relative mRNA expression levels of core genes of SONFH-triggered synovial lesions in the hip synovial tissues were detected by using real-time quantitative PCR(RT-qPCR),and the relative protein expression levels of core genes of SONFH-triggered synovial lesions in the hip synovial tissues were detected by employing Western blotting and immunohistochemical staining,respectively.Additionally,the target cells of SONFH-triggered synovial lesions were detected by immunofluorescence staining.③Cell validation.The rat synovial fibroblasts(SFs)were cultured to construct a synovitis-cell model,and the expression of core genes of SONFH-triggered synovial lesions in blank cells(blank cell group)and synovitis model cells(model cell group)were detected by using RT-qPCR and Western blotting,respectively.Results:①The results of RNA-seq and bioinformatics analysis.One thousand and one SONFH-triggered hip synovial lesions-associated genes were screened out by analysis on differentially expressed genes(DEGs)in hip synovial tissues from different patients,and they were all related to immune response and exosomes,with interferon regulatory factor(IRF)4 and IRF7 identified as the core genes for SONFH-triggered hip synovial lesions,both of which were the key transcription fac-tors in participating in type I interferon responses.②The results of observation on morphology of synovial tissues.The result of HE staining showed that,in FNF group,the hip synovial tissues presented with a normal morphology,without cell proliferation,hypertrophy,or interstitial edema;the hip synovial tissues in HOA group exhibited cell proliferation,with a small amount of neovascularization and cell aggregation;while the marked changes,manifesting as a significant increase in cell proliferation and aggregation,with the neovascularization,were ob-served in hip synovial tissues of SONFH group.③The results of detection on the expression of core genes of SONFH-triggered synovial le-sions in synovial tissues.The relative mRNA expression levels of IRF4,IRF7 and interferon-α(IFN-α)in hip synovial tissues were higher in SONFH group compared to FNF group and HOA group(P=0.000,P=0.000,P=0.000;P=0.001,P=0.000,P=0.036),and were higher in HOA group compared to FNF group(P=0.000,P=0.000,P=0.000).The IRF4,IRF7 and IFN-α proteins were highly ex-pressed in the hip synovial tissues in SONFH group.The relative protein expression levels of IRF4,IRF7 and IFN-α in hip synovial tissues were higher in SONFH group compared to FNF group and HOA group(P=0.001,P=0.000,P=0.000;P=0.000,P=0.014,P=0.000),and were higher in HOA group compared to FNF group(P=0.002,P=0.005,P=0.000).④The results of detection on target cells of SONFH-triggered synovial lesions.The relative protein expression levels of IRF7 and cadherin-11(CDH-11)in hip synovial tissues were higher in SONFH group compared to FNF group and HOA group(P=0.001,P=0.000;P=0.000,P=0.001),and were higher in HOA group compared to FNF group(P=0.001,P=0.000).The SFs were identified as the target cells of SONFH-triggered synovial le-sions.⑤The results of cell verification.The relative mRNA and protein expression levels of IRF4,IRF7,and IFN-α were higher in model cell group compared to blank cell group(P=0.001,P=0.002,P=0.000;P=0.001,P=0.007,P=0.000).Conclusion:The synovial lesions are closely related to immune-inflammatory responses in patients with SONFH.The SFs may be the target cells of SONFH-triggered synovial lesions,with IRF4 and IRF7 as its potential targets.
femur head necrosisglucocorticoidssynovitisRNA-seqfibroblastsinterferon type Ⅰ
万方数据
维普
