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目的观察清络通痹方(Qingluo Tongbi Compound,QTC)对佐剂性关节炎(adjuvant induced arthritis,AIA)大鼠骨破坏相关miRNA表达的影响,探讨其治疗类风湿关节炎的机制。方法建立AIA大鼠滑膜成纤维细胞和单核细胞共培养诱生破骨样细胞模型,应用miRNA芯片的方法初步筛选破骨细胞分化后期miRNA的异常表达谱,并采用实时定量PCR(RT-PCR)对芯片结果进行验证。制备QTC含药血清和空白血清,将破骨细胞随机分为空白组、空白血清组及QTC组,采用RT-PCR检测QTC对差异表达miRNA的影响,并应用生物信息学软件对关键性miRNA进行分析。结果与单核细胞比较,破骨样细胞分化过程中存在明显差异表达的miRNAs共211个,其中表达上调88个,表达下调123个。RT-PCR验证结果与芯片检测结果表达趋势一致。RT-PCR结果显示,QTC干预后miR-140-5p表达明显上调,生物信息学分析结果显示miR-140-5p靶基因显著富集在肌动蛋白细胞骨架的调节、Ras信号通路、c AMP信号通路和Rap1信号通路等信号通路上。结论破骨样细胞分化后期存在多种miRNAs失调,QTC可能通过影响miR-140-5p的表达参与调控破骨细胞的分化。
Objective To investigate the effect of Qingluo Tongbi Compound (QTC) on the expression of miRNAs related to bone destruction in adjuvant induced arthritis (AIA) rats and to explore the mechanism of its treatment for rheumatoid arthritis. Methods The osteoclast-like cells induced by co-culture of synovial fibroblasts and monocytes of AIA rats were established. The miRNA microarray method was used to screen the abnormal expression profiles of miRNAs at the late stage of osteoclast differentiation. Real-time quantitative PCR (RT- PCR) to verify chip results. The QTC-containing serum and blank serum were prepared. The osteoclasts were randomly divided into blank group, blank serum group and QTC group. RT-PCR was used to detect the effect of QTC on differentially expressed miRNA. Bioinformatics software was used to detect the critical miRNA analysis. Results Compared with monocytes, there were 211 differentially expressed miRNAs in osteoclast-like cells, of which 88 were up-regulated and 123 down-regulated. The result of RT-PCR was consistent with the result of chip test. The results of RT-PCR showed that the expression of miR-140-5p was significantly up-regulated after QTC intervention. The results of bioinformatics analysis showed that miR-140-5p target gene was significantly enriched in actin cytoskeleton regulation, Ras signaling pathway, c AMP signal Pathways and Rap1 signaling pathways and other signaling pathways. Conclusion There are many miRNAs imbalance in osteoclast-like cell differentiation stage. QTC may be involved in the regulation of osteoclast differentiation by affecting the expression of miR-140-5p.