变应性哮喘鼠脾巨噬细胞Toll样受体的表达与辅助性T淋巴细胞分泌细胞因子的相关性研究

The study of correlation between the mRNA expression of Toll-like receptors and cytokines of intracellular CD_4^+ T cell in splenic macrophages of allergic asthma mice model

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 understand the expression of mRNA for two Toll-like receptors （TLRs） （ TLR2 and TLR4 ） and two T helper （ Th ） 1 and Th2 cytokines [ interleukin （ IL ） -4, interferon （ IFN ） - gamma] in splenic macrophages of ovalbumin-induced allergic asthma mice model. Methods The expression of mRNA for TLR2, TLR4 and the house-keeping gene HPRT in both splenic macrophages of BALB/c allergic asthma mice and the control mice were analysed by real-time polymerase chain reaction （PCR）. We examined the threshold cycle times （Ct） of eDNA of TLR2 ,TLR4 and HPRT gene to get Ct2 Ct4 and CtH respectively. The value of relative quantity of mRNA level was calculated by the ratio of Ct2/ CtH,Ct4/CtH. After stimulating cells with PMA and Ionomycin with anti-CD3/CD8 in the presence of Brefeldin A, intracellular levels of Thl cytokines （ IFN-γ） and Th2 cytokines （IL-4） were determined for CD3^＋ CD8^- and CD3^＋ CD8^＋ T cells. Results We observed Ct2/CtH （ 1.26 ± 0.04,1.35 ± 0. 11, P 〈 0.01 ） and Ct4/CtH （0. 98 ± 0. 06,1.01 ± 0. 04, P 〉 0. 05） in splenic maerophages between the control mice and allergic asthma mice ; the expression of TLR2 mRNA of allergic asthma mice was significantly reduced. We examined IFN-γ,[IFN-γ,（20. 83 ＋±1.32）% ,（31.78 ±2. 54） %, IL-4（2. 04 ±0. 24）%, （5.91±0. 83）%P 〈0. 05 and P 〈 0. 001 ] , IFN-γ/IL-4 10. 30 ± 1.05,3.58 ± 0. 39 （ P 〈 0.05 ） in splenic maerophages between the control and allergic asthma mice. Conclusions There is no change of the expression of TLR4 mRNA in allergic asthma mice. The expression of TLR2 mRNA in allergic asthma mice is reduced. There has correlation between the expression of TLR2 mRNA and eytokines of intraeellular CD4^＋ T cell in splenic maerophages of allergic asthma mice model;but the reason need further to research.