摘要
背景:既往研究大多关注类风湿关节炎发病过程中的免疫因素,近年来生物力学因素在类风湿关节炎疾病发生和发展中的作用也越来越引起人们的重视。目的:探究Piezo1通过MAPK/ERK5信号通路介导类风湿关节炎成纤维样滑膜细胞凋亡的相关机制。方法:采用组织块法分离培养类风湿关节炎滑膜细胞,利用Flexcell 5000T细胞牵张应力系统构建体外细胞牵张应力模型,构建Piezo1 siRNA基因干扰载体和过表达质粒,根据预实验结果及处理方案,将类风湿关节炎来源滑膜细胞分成6组,即siRNA干扰组、过表达质粒组、空白对照组、siRNA干扰+BIX02188组、过表达质粒+BIX02188组、空白对照+BIX02188组。采用RT-PCR检测Piezo1、ERK5和凋亡相关基因的表达,Fluo-3 AM探针检测细胞内钙离子含量,AV-PI试剂盒检测细胞凋亡水平。结果与结论:①siRNA干扰+BIX02188组细胞内钙离子含量要明显低于siRNA干扰组(P<0.05);过表达质粒+BIX02188组细胞内钙离子含量要明显低于过表达质粒组(P<0.05);②siRNA干扰组的Piezo1、ERK5 mRNA相对表达量要明显低于空白对照组(P<0.05),过表达质粒组明显高于空白对照组(P<0.05);MAPK/ERK5抑制剂BIX02188处理后,Piezo1 mRNA的表达量无明显变化,但是siRNA干扰+BIX02188组的ERK5 mRNA相对表达量要明显低于siRNA干扰组(P<0.05),过表达质粒+BIX02188组的ERK5 mRNA相对表达量要明显低于过表达质粒组(P<0.05);③siRNA干扰组的细胞早期凋亡率、晚期凋亡率和总凋亡率要明显低于空白对照组(P<0.05);过表达质粒组明显高于空白对照组(P<0.05);④结果表明,Piezo1蛋白的过度表达可以促进类风湿关节炎成纤维样滑膜细胞凋亡,并且促凋亡信号通过MAPK/ERK5信号通路介导,可以作为类风湿关节炎治疗的潜在基因靶点。
BACKGROUND:Previous studies have mostly focused on immune factors involved in the pathogenesis of rheumatoid arthritis.Recently,increasing attention has been paid to the role of biomechanical factors in the occurrence and development of rheumatoid arthritis.OBJECTIVE:To explore the mechanism by which Piezo1 mediates apoptosis of fibroblast-like synovial cells in rheumatoid arthritis through MAPK/ERK5 signaling pathway.METHODS:The tissue block method was used to culture synovial cells of rheumatoid arthritis.The stretch stress model of cells in vitro was constructed by Flexcell 5000T cell stretch stress system,and piezo1 siRNA gene interference vector and overexpression plasmid were constructed.According to the preliminary experimental results and treatment plan,fibroblast-like synovial cells of rheumatoid arthritis were divided into six groups:siRNA interference group,overexpression plasmid group,blank control group,siRNA interference+BIX02188 group,overexpression plasmid+BIX02188 group,and blank control+BIX02188 group.The expression of Piezo1,ERK5 and apoptosis-associated genes were detected by RT-PCR,the intracellular calcium content was detected by Fluo-3 AM probe,and the apoptotic level was detected by AV-PI kit.RESULTS AND CONCLUSION:The intracellular Ca2+content of the siRNA interference+BIX02188 group was significantly lower than that of the siRNA interference group(P<0.05);the intracellular Ca2+content of the overexpression plasmid+BIX02188 group was significantly lower than that of the overexpression plasmid group(P<0.05).The relative expression of Piezo1 and ERK5 mRNA in the siRNA interference group was significantly lower than that in the blank control group(P<0.05),and that in the overexpression plasmid group was significantly higher than that in the blank control group(P<0.05).The expression of Piezo1 mRNA did not change after BIX02188 inhibition;however,the relative expression of ERK5 mRNA in the siRNA interference+BIX02188 group was significantly lower than that in the siRNA interference group(P<0.
作者
曲向阳
宋钦勇
Qu Xiangyang;Song Qinyong(Department of Orthopaedic Trauma,Yantai Affiliated Hospital of Binzhou Medical University,Yantai 264100,Shandong Province,China;Department of Spinal Surgery,Yantai Affiliated Hospital of Binzhou Medical University,Yantai 264100,Shandong Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2021年第26期4156-4161,共6页
Chinese Journal of Tissue Engineering Research
关键词
滑膜细胞
类风湿关节炎
机械敏感性离子通道
凋亡
钙离子
通路
牵张应力
synovial cells
rheumatoid arthritis
mechanical sensitive ion channels
apoptosis
calcium ion
pathway
stretch stress
