巨噬细胞中钙调蛋白2的缺失可缓解小鼠炎症性关节炎  

Calmodulin 2 deletion in macrophages alleviates inflammatory arthritis in mice

作　　者：王硕 白阳 WANG Shuo;BAI Yang(Department of Laboratory,Changzhou Second People's Hospital,Changzhou 213164,China)

机构地区：[1]常州市第二人民医院检验科,213164

出　　处：《免疫学杂志》2022年第2期100-107,共8页Immunological Journal

摘　　要：目的研究探讨抑制巨噬细胞中钙调蛋白2的表达治疗炎症性关节炎的作用和相关机制。方法取野生型小鼠(wild type,WT)和钙调蛋白2敲除(knock out,KO)小鼠构建胶原诱导炎症性关节炎模型(collagen induced arthritis,CIA),检测炎症相关因子TNF-α、IL-1β、IL-6和TGF-β的mRNA及蛋白表达变化、小鼠足趾肿胀容积变化以及炎症组织细胞大小和增殖变化。进一步在293T细胞中检测钙调蛋白2和CCL3(macrophage inflammatory protein 1-α,MIP-1α)的结合情况。在骨髓单核巨噬细胞(bone marrow macrophages,BMMs)细胞中转染钙调蛋白2过表达和抑制慢病毒,检测钙调蛋白2潜在下游CCL3的表达。最后,使用抑制钙调蛋白2的慢病毒进行炎症性关节炎小鼠的治疗作用探索,进行上述病理性检测。结果荧光定量PCR和免疫印迹实验显示,钙调蛋白2 KO小鼠关节炎组织炎症因子分泌明显降低;足趾肿胀容积明显减小;炎症组织细胞明显变大,细胞增殖明显增多。荧光素酶实验和染色质免疫共沉淀实验结果显示,钙调蛋白2与野生型CCL3的启动子区域存在结合;进一步,免疫印迹实验显示,关节炎小鼠骨髓巨噬细胞中钙调蛋白2与CCL3间存在负向调控作用。结论给小鼠关节炎组织注射Sh-calmodulin 2慢病毒的治疗作用说明抑制钙调蛋白2的表达可治疗炎症性关节炎。This study was designed to investigate the effect and related mechanism of JAK in the treatment of inflammatory arthritis by inhibiting calmodulin 2 expression in macrophages.Collagen induced arthritis(CIA)model was constructed by using wild type(WT)mice and calmodulin 2 KO mice.The changes of mRNA and protein expression of inflammatory factors TNF-α,IL-1β,IL-6 and TGF-βwere detected by RT-PCR and Western blotting;The changes of swelling volume of toes were detected by toe swelling meter;the changes of cell size and proliferation in inflammatory tissue were detected by HE and EdU.The binding between calmodulin 2 and CCL3 was detected in 293 T cells by Luciferase assay and chromatin immunoprecipitation.Bone marrow macrophages(BMMs)cells were transfected with calmodulin 2 overexpression or suppressed lentivirus,and the expression of potential downstream CCL3 was detected by Western blotting.The pathologic tests were performed to explore the therapeutic effects of calmodulin-2-inhibited lentiviruses in mice with inflammatory arthritis.Fluorescence quantitative PCR and Western blot assay showed that the secretion of inflammatory factors in arthritic tissue of calmodulin 2 KO mice was significantly decreased,and the swelling volume of toes decreased significantly,the inflammatory tissue cells became larger and the cell proliferation increased significantly.Luciferase assay and chromatin immunoprecipitation assay showed that calmodulin 2 was bound to the promoter region of wild-type CCL3.Western blot assay showed that calmodulin 2 and CCL3 were negatively regulated in BMMs of arthritic mice.The therapeutic effect of sh-calmodulin 2 lenti virus injected into arthritic mouse tissues suggests that inhibiting calmodulin 2 expression can treat inflammatory arthritis.

关 键 词：巨噬细胞 钙调蛋白2 炎症性关节炎 

分 类 号：R392.9[医药卫生—免疫学]