目的 探讨急性哮喘小鼠CD4＋T淋巴细胞（简称T细胞）中细胞毒T细胞相关抗原（CTLA）-4与气道重塑的相关性.方法 采用腹腔注射含0.1％卵蛋白（OVA）的Al（OH）3溶液致敏制作急性哮喘小鼠模型,通过雾化吸入2.5％OVA激发哮喘,并使用地塞米松进行干预.最后1次激发完成后48 h,分别采集对照组和哮喘组小鼠眼球血检测IL-4和IFN-γ水平,取肺脏组织行HE染色.分别于最后1次激发完成后6h、12h、24h处死小鼠,采用流式细胞仪检测CD4＋T细胞中CTLA-4的表达水平,同时取肺脏组织行Masson染色检测气道上皮下胶原蛋白的沉积.结果哮喘组小鼠血清IL-4水平高于对照组,IFN-γ水平低于对照组（P〈0.01）.与对照组比较,哮喘组最后1次激发完成后12 h、24 h CD4＋T细胞膜内CTLA-4水平明显升高（P〈0.01）;而地塞米松组最后1次激发完成后6 h、12 h、24 h CD4＋T细胞膜内CTLA-4均明显升高（P〈0.01）.与对照组比较,哮喘组最后1次激发完成后24 h CD4＋T细胞膜外CTLA-4水平明显升高（P〈0.01）;而地塞米松组最后1次激发完成后12 h、24 h CD4＋T细胞膜外CTLA-4均明显升高（P〈0.01）.地塞米松组小鼠最后1次激发完成后12 h、24 h气道上皮下胶原蛋白沉积减少及基底膜增厚较哮喘组缓解.结论 地塞米松干预急性哮喘小鼠后CD4 ＋T细胞中CTLA-4表达明显上调,气道重塑较前减轻.
Background The transcription factor, repressor of GATA-3 （ROG）, can simultaneously suppress the expression of T helper cells （Thl and Th2） cytokines. Since the suppression of Th2 cytokines by GATA-3 is well understood, it is postulated that there are other molecular targets of ROG that can suppress the expression of the Thl cytokines. We hypothesized that ROG might suppress the stimulators of T lymphocyte cytokines such as CD3, CD28, and inducible costimulator （ICOS）, or indirectly enhance the expression of cytokine suppressors such as T lymphocyte-associated antigen-4 （CTLA-4） and CD45. The objective of this study was to clarify the molecular targets of ROG involved in suppressing Thl or Th2 cytokines. Methods Real-time quantitative PCR （RT-PCR） and Western blotting were performed to evaluate the mRNA and protein levels of CD3, CD28, ICOS, CTLA-4, and CD45 in Thl and Th2 cells during various levels of ROG expression. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of interferon-y （IFN-y） and intedeukin （IL）-4 in culture media of Thl and Th2 cells. Results The results showed that the mRNA and protein levels of ROG were relatively low in Thl and Th2 cells （P 〈0.01）. After ROG-pcDNA3.1 transfection, the mRNA and protein level of ROG was significantly elevated, while the expression of ICOS, IFN-y, and IL-4 was markedly down-regulated （P 〈0.01）. Conversely, transfection of ROG-siRNA led to inhibition of ROG expression and up-regulation of ICOS, IFN-y and IL-4 （P 〈0.01）. However, the expression levels of CD3, CD28, CTLA-4 and CD45 did not change in either ROG-pcDNA3.1 or ROG-siRNA-transfected Thl and Th2 cells （P 〉0.05）. Conclusion It is concluded that ROG can inhibit the expression of Thl and Th2 cytokines by down-regulating the expression of ICOS, which might be a potential molecular target for asthma treatment.
