【作者】 黄海；

【导师】 施培华；

【作者基本信息】 浙江大学 ， 临床医学（专业学位）， 2020， 硕士

【摘要】 目的:骨关节炎是老年人最常见的骨科疾病之一。然而,其发病机制尚不明确。本研究旨在研究EGR1在骨关节炎疾病进展中的作用及机制。方法:该研究首先通过荧光定量PCR、蛋白质印迹法、免疫组化等实验检测了EGR1在骨关节炎患者、内侧半月板不稳定(DMM)诱导的骨关节炎及20月龄小鼠的关节软骨样本中的相对表达含量。通过蛋白质印迹法、荧光定量PCR、免疫荧光、茜素红染色等方法研究了EGR1在软骨细胞肥大、软骨基质降解中的作用。通过免疫沉淀、番红固绿染色、免疫组化等方法研究了EGR1发挥作用的机制。通过DMM诱导骨关节炎模型研究了EGR1的抑制剂ML264在骨关节炎发展中的保护作用。结果:EGR1在骨关节炎患者(正常患者作为对照)、DMM诱导的骨关节炎小鼠(Sham组小鼠作为对照)及老年小鼠(年轻小鼠作为对照)的关节软骨样本中表达量显著上调。体外实验表明,IL-1β可以显著上调小鼠软骨原代细胞的EGR1表达。EGR1过表达可以抑制COl2A1的表达,提高基质金属蛋白酶的表达,并导致软骨细胞肥大。而使用RNAi沉默EGR1则有相反的效果。我们研究了潜在机制,发现EGR1过表达促使其与Kruppel-like Factor 5(KLF5)蛋白结合,从而抑制了KLF5的泛素化降解过程,导致KLF5蛋白水平的上升。此外,EGR1促使β-catenin核转移从而调控软骨细胞肥大。我们还发现EGR1的抑制剂ML264在体外实验中能抑制IL-1β诱导的软骨基质降解,在体内实验中能抑制DMM诱导的骨关节炎。结论:EGR1通过激活KLF5和β-catenin通路促进软骨细胞肥大退变。更多还原

【Abstract】 Objective : Osteoarthritis is one of the most common orthopedic diseases in the elderly.However,its pathogenesis remains unclear.This study aimed to explore the role and mechanism of EGR1 in the progression of osteoarthritis.Methods:In this study,the relative expression of EGR1 in articular cartilage samples of osteoarthritis patients,destabilized medial meniscus(DMM)-induced osteoarthritis and 20-month-old mice were firstly detected by quantitative PCR,western blot and immunohistochemistry.The effects of EGR1 on chondrocyte hypertrophy and cartilage matrix degradation were studied by western blot,quantitative PCR,immunofluorescence and alizarin red staining.The mechanism of how EGR1 works was studied by immunoprecipitation,safranin-O-fast green staining and immunohistochemistry.The protective effect of ML264,an inhibitor of EGR1,on the development of osteoarthritis was studied in DMM-induced osteoarthritis model.Results:EGR1 expression was significantly up-regulated in articular cartilage samples from osteoarthritis patients(normal patients as control),DMM-induced osteoarthritismice(Sham group as control)and elderly mice(young mice as control).In vitro experiments indicated that IL-1β could significantly enhance EGR1 expression in primary mouse chondrocytes.EGR1 over-expression could inhibit COl2A1 expression,enhance matrix metalloproteinase expression,resulting in chondrocyte hypertrophy.Silencing EGR1,using RNAi,had the opposite effects.We investigated the underlying mechanism and found that EGR1 over-expression promoted its integration with Kruppel-like Factor 5(KLF5)protein,which inhibited the ubiquitination degradation process of KLF5 and led to the increase of KLF5 protein level.Moreover,EGR1 prompted β-catenin nuclear transportation to control chondrocyte hypertrophy.We also found that the EGR1 inhibitor,ML264,protected chondrocytes from IL-1β-mediated cartilage matrix degradation in vitro and DMM-induced osteoarthritis in vivo.Conclusion : EGR1 promotes the cartilage degeneration and hypertrophy by activating the Kruppel-like Factor 5 and β-catenin signaling.更多还原