阻断B7/CD28通路对类风湿关节炎小鼠的影响  

The effect of blocking B7/CD28 pathway on micewith rheumatoid arthritis

作　　者：刘颖[1] 黄艳艳[1] 詹宇威 詹锋[1] Liu Ying;Huang Yanyan;Zhan Yuwei;Zhan Feng(Dept of Rheumatology and Immunology,Hainan Provincial People′s Hospital(Hainan Hospital Affiliated to Hainan Medical College),Haikou 570000)

机构地区：[1]海南省人民医院(海南医学院附属海南医院)风湿免疫科,海口570000

出　　处：《安徽医科大学学报》2022年第4期515-522,共8页Acta Universitatis Medicinalis Anhui

基　　金：海南省自然科学基金青年基金项目(编号:819QN347)。

摘　　要：目的探讨阻断B7/CD28通路对类风湿关节炎小鼠的影响及可能机制。方法40只DBA/1小鼠随机分正常对照组、模型组、细胞毒性淋巴细胞抗原4免疫球蛋白(CTLA4-Ig)低剂量组和CTLA4-Ig高剂量组,除正常对照组外,其余三组小鼠均制备类风湿关节炎模型。在二次免疫结束后第0、5、10天CTLA4-Ig低、高剂量组分别腹腔注射50、100 mg/kg CTLA4-Ig,正常对照组和模型组注射等量生理盐水。游标卡尺测定小鼠同侧后足掌厚度,进行关节炎评分;ELISA法检测血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)和白细胞介素-17(IL-17)的水平,Micro-CT扫描采集图像并测量骨组织形态计量学参数,HE染色观察膝关节病理形态变化,TRAP染色检测膝关节组织内破骨细胞,实时荧光定量PCR和Western blot检测骨保护素(OPG)、核因子κB受体活化因子(RANK)、核因子κB受体活化因子配体(RANKL)mRNA及蛋白表达情况,免疫组织化学染色检测核因子κB(NF-κB)表达,Western blot检测NF-κB信号通路相关蛋白表达。结果与模型组比较,经过CTLA4-Ig高剂量治疗后,RA小鼠足肿胀和关节炎评分均降低,血清中TNF-α、IL-1β、IL-6及IL-17水平均降低,膝关节受损程度减小,骨密度(BMD)、骨小梁厚度(Tb.Th)、骨体积(BV)及骨体积分数(BV/TV)升高,抗酒石酸酸性磷酸酶(TRAP)阳性细胞数目减少,组织内OPG mRNA与蛋白相对表达量上调,而RANK、RANKL mRNA与蛋白相对表达量均下调,同时,p-p65、p-IκBα蛋白相对表达量降低,NF-κB阳性表达面积减少,差异有统计学意义(P<0.01);通过CTLA4-Ig低剂量治疗后,RA小鼠血清中TNF-α、IL-1β、IL-6以及IL-17水平较模型组下降,组织内NF-κB阳性表达面积减少,差异有统计学意义(P<0.01)。结论CTLA4-Ig阻断B7/CD28通路,对小鼠类风湿关节炎具有明显的治疗作用,可抑制炎性反应,减少骨破坏,并阻止NF-κB信号途径激活。Objective To investigate the effect of blocking the B7/CD28 pathway on rheumatoid arthritis mice and its possible mechanism.Methods 40 DBA/1 mice were randomly divided into normal control group,model group,CTLA4-Ig low-dose group and cytotoxic T lymphocyte antigen-4 immunolobulin(CTLA4-Ig)high-dose group.Except for the normal control group,the other three groups were prepared for rheumatoid arthritis models.At 0,5 and 10 days after the second immunization,the CTLA4-Ig low-dose group and CTLA4-Ig high-dose group were intraperitoneally injected with 50 mg/kg and 100 mg/kg CTLA4-Ig,respectively,the normal control group and the model group were injected with the same amount of saline.Vernier calipers were used to measure the thickness of the ipsilateral hindfoot of the mouse for arthritis score;the serum levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)and interleukin-17(IL-17)were detected by ELISA,images were acquired and bone tissue morphometric parameters were measured through Micro-CT scanning,HE staining observed the pathological changes of the knee joint,TRAP staining detected osteoclasts in knee joint tissues,real-time fluorescent quantitative PCR and Western blot detected osteoprotegerin(OPG),receptor activator of NF-κB(RANK),receptor activator of NF-κB ligand(RANKL)mRNA and protein expression,immunohistochemical staining detected nuclear factorκB(NF-κB)expression.Western blot was used to detect the expression of related proteins in the NF-κB signaling pathway.Results Compared with the model group,after high-dose CTLA4-Ig treatment,RA mouse foot swelling and arthritis scores were reduced,the levels of TNF-α,IL-1β,IL-6 and IL-17 in serum were reduced,the knee joint damage was significantly reduced,bone mineral density(BMD),trabecular thickness(Tb.Th),bonevolume(BV)and bone volume fraction(BV/TV)increased,the number of TRAP positive cells decreased,the relative expression of OPG mRNA and protein in the tissue was up-regulated,while the relative expression of RANK,RAN

关 键 词：类风湿关节炎 CTLA4-IG B7/CD28共刺激通路 炎症 骨破坏 

分 类 号：R593.22[医药卫生—内科学]