激素性股骨头坏死滑膜病变分子机制的实验研究  

Molecular mechanisms of synovial lesions in steroid-induced osteonecrosis of the femoral head:an experimental study

作　　者：方伟华 庄至坤 彭鹏 肖方骏 杨晓强 林锟 何伟 魏秋实 何敏聪 FANG Weihua;ZHUANG Zhikun;PENG Peng;XIAO Fangjun;YANG Xiaoqiang;LIN Kun;HE Wei;WEI Qiu-shi;HE Mincong(The Third Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510006,Guangdong,China;Quanzhou Orthopedic-Traumatological Hospital,Quanzhou 362000,Fujian,China;The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510378,Guangdong,China)

机构地区：[1]广州中医药大学第三临床医学院,广东广州510006 [2]泉州市正骨医院,福建泉州362000 [3]广州中医药大学第三附属医院,广东广州510378

出　　处：《中医正骨》2024年第9期49-58,75,共11页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：国家自然科学基金项目(82274544,82004392);广东省基础与应用基础研究基金项目(2023A1515010551);国家中医药管理局高水平中医药重点学科建设项目(zyyzdxk-2023106);福建中医药大学中医骨伤科学学科开放课题资助项目(XGS2023008);毕节市科学技术局2022年度“揭榜挂帅”项目(毕科合重大专项[2022]1号);广东省中医骨伤研究院开放课题重点项目(GYH202101-01,GYH202101-04)。

摘　　要：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�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 arthroplasty for RNA-seq and bioinformatics analysis to screen the core genes of synovial lesions in SONFH.②Synovial tissues detection.The hip synovial 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 factors in participating in type I interferon resp

关 键 词：股骨头坏死 糖皮质激素 滑膜炎 RNA测序 成纤维细胞 干扰素Ⅰ型 

分 类 号：R681.8[医药卫生—骨科学]