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目的研究卵清蛋白诱导的变应性哮喘(简称哮喘)模型鼠脾脏巨噬细胞Toll样受体(TLR)中TLR2和TLR4 mRNA的表达与该组织内辅助性T淋巴细胞(CD4+T)分泌的细胞因子的相互关系。方法用实时荧光聚合酶链反应(FQ-PCR)检测TLR2和TLR4 mRNA与次黄嘌呤磷酸核糖基转移酶管家基因(hypoxanthine-phosphoribosyl-transferase gene,HPRT)cDNA的荧光域值循环数(Ct),分别记为Ct2、Ct4和CtH,计算Ct2、Ct4与CtH的比值(Ct2／CtH、Ct4／CtH)为标本的TLR2和TLR4在mRNA水平上的相对表达量。进一步采用双标记流式细胞分析技术检测鼠脾脏CD4+T淋巴细胞,随后将单克隆抗体和高尔基体转运阻断剂结合,检测CD4+T淋巴细胞胞内γ-干扰素(IFN-γ)和白细胞介素-4(IL-4)的变化。结果对照组与哮喘组小鼠Ct2／CtH差异有统计学意义(分别为1．26±0．04,1．35±0．11,P<0．01),哮喘组TLR2 mRNA表达下调;对照组与哮喘组小鼠Ct4／CtH差异无统计学意义(分别为0．98±0．06,1．01±0．04,P>0．05);对照组与哮喘组小鼠IFN-γ和IL-4的变化差异有统计学意义[IFN-γ分别为(20．83±1．32)％,(31．78±2．54)％;IL-4分别为(2．04±0．24)％, (5．91±0．83)％,P<0．05和P<0．01],哮喘组脾脏CD4+T淋巴细胞IFN-γ和IL-4分泌增加。对照组与哮喘组小鼠IFN-γ/IL-4的比值差异有统计学意义(分别为10．30±1．05,3．58±0．39,P<0．05)。结论变应性哮喘模型鼠脾脏巨噬细胞TLR4 mRNA的表达无变化,但TLR2 mRNA的表达下调;在哮喘模型鼠脾脏CD4+T淋巴细胞中IFN-γ/IL-4比值降低。TLR2 mRNA表达的下调与IFN-γ和IL-4分泌的变化存在相关性,其机理有待进一步深入研究。
Objective To study the expression of TLR2 and TLR4 mRNA in Toll-like receptor (TLR) of spleen macrophages induced by ovalbumin in allergic asthma model rats and the secretion of T helper T lymphocytes (CD4 + T) The Relationship Between Cytokines. Methods Real-time fluorescent polymerase chain reaction (FQ-PCR) was used to detect the number of fluorescence domain values (Ct) of TLR2 and TLR4 mRNA and hypoxanthine-phosphoribosyl-transferase gene (HPRT) (Ct2 / CtH, Ct4 / CtH) were calculated as Ct2, Ct4 and CtH. The relative expression levels of TLR2 and TLR4 at the mRNA level were calculated. Further use of dual-labeled flow cytometry to detect the spleen CD4 + T lymphocytes, and then combine the monoclonal antibody with the Golgi transport blocker to detect the intracellular IFN-γ (IFN-γ) of CD4 + T lymphocytes Interleukin-4 (IL-4) changes. Results There was significant difference in Ct2 / CtH between control group and asthma group (1.26 ± 0.04, 1.35 ± 0.11, P <0.01, respectively), and TLR2 mRNA was down-regulated in asthma group There was no significant difference in Ct4 / CtH between the asthma group and the asthma group (0.98 ± 0.06,1.01 ± 0.04, P> 0.05). The IFN-γ (20.83 ± 1.32)%, (31.78 ± 2.54)%, respectively; IL-4 was (2.04 ± 0) .24%, (5.91 ± 0.83)%, P <0.05 and P <0.01]. The secretion of IFN-γ and IL-4 in CD4 + T lymphocytes of asthmatic group increased. The ratio of IFN-γ / IL-4 in control group and asthma group was significantly different (10.30 ± 1.05,3.58 ± 0.39, P <0.05 respectively). Conclusion The expression of TLR4 mRNA in splenic macrophages of allergic asthma model rats was not changed, but the expression of TLR2 mRNA was down-regulated. The ratio of IFN-γ / IL-4 in CD4 + T lymphocytes of asthmatic mice decreased. The down-regulation of TLR2 mRNA expression correlates with the changes of IFN-γ and IL-4 secretion, and its mechanism remains to be further studied.