小鼠补体调节蛋白对CD4+T淋巴细胞的调控作用及其机制

目的 研究小鼠补体调节蛋白Crry对CD4+T淋巴细胞的调控作用及诱导同种移植免疫低反应性的机制.方法 分离C57BL/6小鼠脾淋巴细胞,用免疫磁珠法分选出CD4+T淋巴细胞后,将CD4+T淋巴细胞分为A、B、C、D、E和F组,分别用抗小鼠CD3、CD28、Crry、CD3/CD28、CD3/Crry和CD3/CD28/Crry抗体共刺激通路与CD4+T淋巴细胞进行反应,采用噻唑蓝(MTT)法检测各组CD4+T淋巴细胞的增殖情况,并采用酶联免疫吸附试验检测CD4+T淋巴细胞培养上清中白细胞介素2(IL-2)、γ干扰素(γ-IFN)、IL-4和IL-10的水平;另外,以BALB/c小鼠和C57BL/6小鼠的脾细胞分别作为刺激细胞和反应细胞,建立同种混合淋巴细胞反应(MLR)体系并加入抗小鼠Crry抗体,通过岍法观察Crry对MLR的影响.结果 D、E、F组的CD4+T淋巴细胞均出现明显增殖,增殖活性显著高于A、B、C组(P<0.05),其中F组显著高于D组和E组(P<0.05),D组和E组间增殖活性的差异无统计学意义.D组CD4+T淋巴细胞经抗CD3/CD28抗体共刺激后,培养上清中γ-IFN和IL-2的水平显著升高,与A、B、C和E组比较,差异均有统计学意义(P<0.05),但与F组的差异无统计学意义;E组CD4+T淋巴细胞经抗CD3/Crry抗体共刺激后,IL-4的水平显著升高,与A、B、C、D组比较,差异均有统计学意义(P<0.05),但显著低于F组(P<0.05);各组间IL-10水平的差异无统计学意义.Crry可以明显抑制MLR中的细胞增殖(P<0.05).结论 补体调节蛋白Crry能刺激CD4+T淋巴细胞的增殖,并使其IL-4的表达升高及抑制IL-2和γ-IFN的表达,从而诱导同种移植免疫低反应性.

Complement regulatory protein Crry regulates CD4+ T lymphocytes and induces allograft immune hyporesponsiveness

Objective To investigate the allograft immune hyporesponsiveness induced by complement regulatory protein Crry as well as CD4+ T lymphocytes involved.Methods CD4+ T cells were isolated from spleens of C57BL/6 mice by magnetic-activated cell sorting, and CD4+ T cell proliferation was induced by anti-CD3, anti-CD28, anti-Crry, co-stimulations of anti-CD3/anti-CD28, anti-CD3/anti-Crry, or the monoclonal antibody panel against CD3/CD28/Crry.Production of interleukin-2 (IL-2), γ-interferon (γ-IFN), interleukin-10 (IL-10) and interleukin-4 (IL-4) was detected in the supernatants of different groups.Allo-MLR was examined by MTT colorimetry with spleen cells from BALB/c mice as the stimulators and spleen cells of C57BL/6 mice as responders.Results Compared with CD3 group, CD3/CD28, CD3/Crry and CD3/CD28/Crry co-stimualtion all could significantly induce more proliferation (P<0.05).CD3/CD28/Crry co-stimulation significantly increased the proliferation compared with CD3/CD28 or CD3/Crry co-stimulation (P<0.05).IL-2 and γ-IFN levels were significantly increased in CD3/CD28 co-stimulation as compared with CD3 or CD28 or Crry or CD3/Crry group (P<0.05).IL-4 levels were significantly increased in CD3/Crry co-stimulation as compared with CD3 or CD28 or Crry or CD3/CD28 groups (P<0.05).There was no significant difference in IL-10 level among the different groups (P>0.05).CD3/Crry co-stimulation significantly inhibited proliferation in mixed lymphocyte culture (P<0.05).Conclusion Complement regulatory protein Crry induces IL-4 in CD4+ T ceils, suppresses IL-2 and γ,-IFN production, and induces allograft immune hyporesponsiveness