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目的分析变应原刺激前后变应性哮喘患儿外周血T淋巴细胞表面协同刺激分子及胞内细胞因子表达的变化,探讨CD28家族不同协同刺激信号在变应性哮喘免疫病理机制中的作用。方法选取尘螨变应性哮喘患儿(哮喘组)和健康儿童(健康对照组)各30例,密度梯度离心法分离其外周血单个核细胞,应用免疫荧光标记和流式细胞术检测尘螨刺激前后体外培养的CD4+T淋巴细胞表面协同刺激分子CD28、可诱导共刺激分子(ICOS)和细胞毒T淋巴细胞相关抗原4(CTLA-4)的表达,运用细胞内染色技术检测CD4+T淋巴细胞内细胞因子γ干扰素(IFN-γ)、IL-4和IL-13的表达。并运用统计学方法比较哮喘组和健康对照组之间的差异。结果哮喘组患儿外周血CD4+T淋巴细胞表面CD28和ICOS的表达与健康对照组比较差异均无统计学意义(Pa>0.05),而CTLA-4的表达显著降低(P<0.01);细胞内IFN-γ表达水平显著升高(P<0.000 1),而IL-4和IL-13表达水平无明显变化(Pa>0.05)。经尘螨刺激后,体外培养的哮喘患儿外周血CD4+T淋巴细胞表面ICOS的表达较健康对照组儿童显著上调(P<0.000 1),CD28和CTLA-4的表达则无明显变化(Pa>0.05);细胞内细胞因子IL-4和IL-13的表达显著上调(Pa<0.000 1),而IFN-γ的表达则无明显差异(P>0.05)。结论变应性哮喘患儿外周血存在组成性的CTLA-4的表达下调和细胞因子IFN-γ的表达上调,介导Th1型细胞的异常活化;而变应原尘螨的刺激又介导了ICOS依赖的Th2型细胞的分化,导致Th1/Th2失衡。
Objective To analyze the changes of costimulatory molecules and intracellular cytokines on T lymphocytes in peripheral blood of children with allergic asthma before and after allergic asthma stimulation and to explore the role of CD28 family of different costimulatory signals in the immunopathogenesis of allergic asthma. Methods Thirty children with allergic asthma (allergic asthma group) and 30 healthy children (healthy control group) were selected respectively. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Immunofluorescence staining and flow cytometry were used to detect dust mite Before and after stimulation, the surface costimulatory molecule CD28 of CD4 + T lymphocytes cultured in vitro could induce the expression of costimulatory molecule (ICOS) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4), and the intracellular staining was used to detect the expression of CD4 + T Lymphocyte cytokine interferon (IFN-γ), IL-4 and IL-13 expression. Statistical methods were used to compare the differences between the asthma group and the healthy control group. Results The expression of CD28 and ICOS on CD4 + T lymphocytes in asthmatic children was not significantly different from that in healthy controls (P> 0.05), while the expression of CTLA-4 was significantly decreased (P <0.01) (P <0.000 1), while the expression levels of IL-4 and IL-13 had no significant change (Pa> 0.05). After dust mite stimulation, the expression of ICOS on CD4 + T lymphocytes in peripheral blood of children with asthma increased significantly (P <0.000 1), while the expression of CD28 and CTLA-4 had no significant change (Pa > 0.05). The intracellular cytokines IL-4 and IL-13 expression were significantly upregulated (Pa0.0001), while the expression of IFN-γ was not significantly different (P> 0.05). Conclusion The expression of CTLA-4 in peripheral blood of children with allergic asthma is down-regulated and the expression of IFN-γ is up-regulated, which leads to the abnormal activation of Th1-type cells. The stimulation of allergen dust mite mediates Differentiation of ICOS-dependent Th2-type cells results in an imbalance of Th1 / Th2.