丙二酸次氮酰胺对刀豆蛋白刺激的胶原性关节炎模型大鼠CD4~+CD25~+调节性T淋巴细胞体外分化的影响

目的:研究来氟米特活性代谢物丙二酸次氮酰胺(A771726)对刀豆蛋白(ConA)刺激的胶原性关节炎(CIA)模型大鼠CD4+CD25+调节性T淋巴细胞(CD4+CD25+Tregs)体外分化的影响及其治疗类风湿性关节炎的作用机制。方法:取大鼠分为正常对照组和CIA模型组,建模后制备脾淋巴细胞悬液,再分为正常组、模型组和A771726低、中、高剂量(0.1、1、10μmol·L-1)组,各组加入ConA刺激和相应药物培养液培养48h后,以MTT法检测各组脾淋巴细胞增殖情况,流式细胞术检测各组CD4+CD25+Tregs占CD4+T淋巴细胞的比例;反转录-聚合酶链式反应检测各组CD4+CD25+Tregs特异性标志物Foxp3和转化生长因子β(TGF-β)mRNA表达情况;酶联免疫吸附测定法检测各组TGF-β浓度。结果:与正常组比较,模型组脾淋巴细胞增殖明显升高(P<0.01),CD4+CD25+Tregs占CD4+T淋巴细胞的比例、Foxp3和TGF-βmRNA表达、TGF-β浓度均明显降低(P<0.05);与模型组比较,A7717263个剂量组脾淋巴细胞增殖明显降低(P<0.05或P<0.01),A771726高剂量组CD4+CD25+Tregs占CD4+T淋巴细胞的比例明显升高,A771726中、高剂量组Foxp3和TGF-βmRNA表达、TGF-β浓度均明显升高(P<0.05或P<0.01)。结论:A771726可抑制CIA模型大鼠脾淋巴细胞增殖,诱导CD4+CD25+Tregs的分化,其机制可能与上调TGF-β的表达和分泌有关。

Effect of Malonic Acid Amide Nitrogen on the Differentiation of CD4~+CD25~+ Tregs of ConA-induced CIA Rats in Vitro

OBJECTIVE:To study the effect and mechanism of active metabolite malonic acid amide nitrogen(A771726) on the differentiation of CD4+CD25+ Tregs from collagen-induced arthritis(CIA) rats in vitro.METHODS:Rats were divided into normal control group and CIA model group.The splenocyte suspension was prepared after modeling then divided into normal group,model group and A771726 low-dose,medium-dose and high-dose groups(0.1、1、10 μmol·L-1).Those groups were cultured in culture solution of ConA and relevant medicine for 48 h.The proliferation of spleen lymphocytes from CIA rats was checked with MTT assay;immunofluorescence flow cytometry was used to detect the proportion of CD4+CD25+ Tregs in CD4+T;RT-PCR was used to detect the expression of Foxp3 mRNA and TGF-β mRNA;ELISA was used to detect the content of TGF-β.RESULTS:Compared with normal group,the proliferation of ConA-induced splenocytes of CIA rats in model group increased significantly(P<0.01),while the proportion of CD4+CD25+ Tregs,the expression of Foxp3 and TGF-β mRNA and the content of TGF-β decreased significantly(P<0.05).Compared with model group,the proliferation of ConA-induced splenocytes in A771726 groups decreased significantly(P<0.05 or P<0.01),while the proportion of CD4+CD25+ Tregs in CD4+T of A771726 high-dose group increased significantly,and the expression of Foxp3 and TGF-β mRNA and the content of TGF-β in A771726 medium-dose and high-dose groups increased significantly(P<0.05 or P<0.01).CONCLUSION:A771726 could inhibit the proliferation of splenocytes of CIA rats,and facilitate the differentiation of CD4+CD25+Tregs,which may be related to the up-regulation of the expression and secretion of TGF-β.