摘要
目的:探索活性氧(ROS)对类风湿性关节炎(RA)骨破坏的影响。方法:建立Ⅱ型胶原诱导性类风湿性关节炎(CIA)大鼠模型,观察大鼠关节红肿畸形等症状;HE和Masson三色染色观察关节滑膜、软骨、骨组织等病理变化;抗酒石酸酸性磷酸酶(TRAP)染色观察关节破骨细胞生成情况;二氢乙锭(DHE)荧光探针检测关节组织中ROS的表达。体外巨噬细胞集落刺激因子(M-CSF)和核因子κB受体活化因子配体(RANKL)诱导小鼠原代骨髓巨噬细胞(BMMs)48 h、96 h后,TRAP和鬼笔环肽染色观察破骨细胞形成和分化情况,RT-PCR和免疫荧光技术检测TRAP、基质金属蛋白酶9(MMP-9)、组织蛋白酶K(CTSK)mRNA和CTSK、MMP-9蛋白的表达;DCFH-DA探针检测破骨细胞中ROS水平变化。结果:CIA大鼠关节组织呈现炎症细胞浸润、滑膜异常增生、TRAP阳性细胞大量形成,骨破坏严重区域ROS表达明显增强。此外,成功构建体外破骨细胞分化模型,48 h开始出现多核破骨细胞,96 h形成大量成熟破骨细胞。溶骨功能相关因子mRNA和蛋白的表达呈诱导时间依赖性增加,同时ROS的表达呈现趋势性累积;而且,ROS抑制剂NAC明显抑制了破骨细胞的分化。结论:ROS可能通过调控破骨细胞的分化促进RA骨破坏,抑制ROS产生可明显抑制破骨细胞分化。
Objective:To study the effect of reactive oxygen species(ROS)on bone destruction of rheumatoid arthritis(RA).Methods:The rat model of collagen-induced rheumatoid arthritis(CIA)was established,then the rats'symptoms of joint swelling and deformity in each group were recorded.The pathological changes of synovium,cartilage and bone were observed by HE and Masson staining.TRAP staining was used to mark mature osteoclasts.The ROS in adjuvant arthritis rats were analyzed by DHE fluorescent.Bone marrow macrophages(BMMs)were treated with M-CSF and RANKL for 48 h,96 h.Formation and activation of osteoclasts were observed by TRAP staining and phalloidin staining.The expression of osteoclast specific genes,TRAP,MMP-9,CTSK mRNA and MMP-9,CTSK proteins levels were detected by RT-PCR and immunofluorescence staining separately.DCFH-DA fluorescence staining was used for intracellular ROS detection.Results:The results of pathological sections showed that CIA rats had arthritic cell infiltration,synovial hyperplasia and a massive formation of TRAP positive cells with the vigorous ROS expression at the same time.And osteoclast differentiation model was successfully constructed in vitro,then multinucleated osteoclast were formed at 48 h and more at 96 h.The expression of ROS in cells showed a tendency accumulation with time as well as the expression of osteoclast related genes under RANKL induction;moreover,after intervention with NAC,a ROS inhibitor,osteoclast differentiation was significantly inhibited.Conclusion:ROS affects RA bone destruction by participating in osteoclast differentiation.Inhibition of ROS production can significantly inhibit osteoclast differentiation.
作者
郭婉怡
袁蓓
张铌雪
孔祥英
苏晓慧(指导)
林娜(指导)
GUO Wan-Yi;YUAN Bei;ZHANG Ni-Xue;KONG Xiang-Ying;SU Xiao-Hui;LIN Na(Institute of Chinese Materia Medica China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2021年第18期2182-2186,2193,共6页
Chinese Journal of Immunology
基金
国家自然科学基金青年科学基金项目(81703787)
国家重点研发计划(2017YFC1702005)
北京市自然科学基金面上项目(7202142)。
关键词
活性氧
破骨细胞
骨髓巨噬细胞
类风湿性关节炎
大鼠
Reactive oxygen species
Osteoclasts
Bone marrow macrophages
Rheumatoid arthritis
Rats
