CD300c-Ig对小鼠关节损伤的影响及机制

Effects of CD300c-Ig on joint injury in mice with collagen-induced arthritis

目的探讨CD300c-Ig对胶原诱导性关节炎(collagen induced arthritish,CIA)小鼠关节损伤的影响及作用机制。方法分别在第1天及第21天免疫DBA/1J小鼠,构建CIA模型,随机分为处理组和对照组,每组10只。处理组腹腔注射20μg CD300c-Ig 100μL,1次/3 d,共6次,总剂量为120μg;对照组则腹腔注射相同质量的对照Ig蛋白(control-Ig),1次/3 d,共6次。观察四肢关节肿胀程度,记录关节炎指数评分;DBA/1J小鼠初次免疫40 d后,取小鼠脾脏制成单细胞悬液,流式细胞术检测调节性T细胞(T regulatory cells,Treg)、辅助性T细胞(T helper cells,Th17)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、干扰素-γ(interferon-γ,IFN-γ)的表达;取小鼠下肢,脱钙,制成切片进行苏木精-伊红染色及番红O染色,观察小鼠关节病理特征并进行评分。采用荧光定量PCR法检测叉头框蛋白P3(forkhead box protein P3,Foxp3)、白介素-17(interleukin-17,IL-17)和白介素-23(interleukin-23,IL-23)的mRNA表达。结果处理组关节肿胀程度较对照组减轻,关节炎指数与对照组相比明显降低;苏木精-伊红染色及番红O染色显示,处理组关节滑膜细胞增殖较对照组减轻、血管翳形成、骨质侵蚀情况均比对照组有明显改善(P<0.05);流式细胞术检测结果显示,处理组脾脏Th17细胞比例较对照组明显降低(P<0.05),Treg细胞比例较对照组明显升高(P<0.05);同时,处理组脾脏TNF-α、IFN-γ表达与对照组相比明显降低(P<0.05);荧光定量PCR结果显示,处理组IL-17和IL-23 mRNA表达水平明显低于对照组。结论CD300c-Ig可减轻CIA小鼠的关节损伤,其机制与其对Th17/Treg细胞平衡的调控和抑制炎性因子的释放有关。

Objective To investigate the effects and mechanism of CD300c-Ig on joint injury in collagen-induced arthritis(CIA)mice.Methods To construct the CIA model,DBA/1J mice were immunized on day 1 and day 21,and then randomly divided into the treatment group and control group,with 10 mice in either group.Mice in the treatment group were intraperitoneally injected with 100μL of 20μg CD300c-Ig once every 3 days for 6 times,with a total dose of 120μg;mice in the control group were intraperitoneally injected with control Ig of the same volume once every 3 days for 6 times.The degree of swelling in the joints of the extremities was observed and the arthritis index score was recorded.Forty days after the initial immunization,the spleens of the mice were taken to make single-cell suspension,and the expressions of T regulatory cells(Treg),T helper cells(Th17),tumor necrosis factor-α(TNF-α),and interferon-γ(IFN-γ)were detected with flow cytometry.The lower limbs of the mice were taken,decalcified,and sliced for hematoxylin-eosin staining and safranin O staining to visualize the pathological features of the joints and to score them.The mRNA expressions of forkhead box protein P3(Foxp3),interleukin-17(IL-17)and interleukin-23(IL-23)were detected with fluorescence quantitative PCR.Results The degree of joint swelling and AI in the treatment group were significantly reduced compared with those in the control group.Hematoxylin-eosin staining and saffron O staining showed that the proliferation of synoviocytes in the synovial joints of the treatment group was reduced,and the formation of vascular cataracts and the erosion of bone were significantly improved(P<0.05).Flow cytometry showed that the proportion of Th17 cells in the spleens of the treatment group was significantly lower(P<0.05)while the proportion of Treg cells was significantly higher(P<0.05).The expressions of TNF-αand IFN-γin the spleen of the treatment group were significantly lower(P<0.05).Fluorescence quantitative PCR showed that the mRNA expressions of IL-17 and IL-23 in the treatment group were significantly lower.Conclusion CD300c-Ig can reduce joint injury in CIA mice,and the mechanism is related to its regulation of Th17/Treg cell balance and inhibition of inflammatory factor release.