论文部分内容阅读
目的:探究肺泡表面活性蛋白A(surfactant protein A,SP-A)能否通过调控滤泡辅助性T细胞(follicular helper T cell,Tfh)的分化参与调节哮喘病理过程。方法:6~8周的雌性野生型(wild-type,WT)及SP-A基因敲除(n Sp-n a-/-)型C57BL/6J小鼠,利用鸡卵白蛋白(ovalbumin,OVA)和氢氧化铝免疫构建哮喘模型(WT哮喘小鼠:8只,n Sp-n a-/-哮喘小鼠:8只),并分别设立磷酸盐缓冲液(phosphate buffer saline,PBS)处理的对照组(WT对照小鼠:6只,n Sp-n a-/-对照小鼠:4只)。HE染色观察小鼠肺部病理改变,流式细胞术检测脾脏和纵隔淋巴结中Tfh细胞及其亚群的组成,Tfh亚群根据其胞内干扰素-γ(interferon-γ,IFN-γ)、白细胞介素(interleukin,IL)-17和IL-4的表达情况,分别定义为IFN-γn +Tfh、IL-17n +Tfh和IL-4n +Tfh细胞。酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)用于检测支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)中免疫球蛋白(immunoglobulin,Ig)E的水平。将T-B细胞共培养,检测培养体系中IgE抗体的水平。在体外,给予人肺泡表面活性蛋白A(human SP-A,hSP-A)处理并设立对照组,培养5 d后检测体系中Tfh亚群的分布情况。n 结果:OVA激发后小鼠BALF中和肺组织内有大量炎症细胞浸润,WT及n Sp-n a-/-哮喘小鼠BALF中炎症细胞总数显著高于其相对应对照组小鼠[(11.38±1.43)×10n 4比(6.40±0.68)×10n 4,(14.38±1.52)×10n 4比(6.25±0.48)×10n 4,n t值分别为2.61和3.64,n P值均<0.05],差异具有统计学意义。另外,OVA激发后,WT和n Sp-n a-/-哮喘小鼠BALF中IgE的水平明显高于其相应的对照组小鼠[(16.85±2.50)pg/mL比(4.94±2.01)pg/mL,(25.52±3.17)pg/mL比(8.05±2.90)pg/mL,n t值分别为3.63和3.58,n P值均<0.05)],且n Sp-n a-/-哮喘小鼠BALF中IgE抗体的水平明显高于WT哮喘小鼠(n t=2.20,n P0.05)。Tfh细胞的亚群组成发生变化,其中IL-4n +Tfh比例与肺泡灌洗液中IgE水平呈正相关(n r=0.50,n P<0.05),而IFN-γn +Tfh比例与肺泡灌洗液中IgE水平呈负相关(n r=-0.56,n P<0.05)。脾脏中IFN-γn +Tfh/IL-4n +Tfh的比值在n Sp-n a-/-哮喘小鼠中显著低于WT小鼠(n t=2.31,n P<0.05),且与肺泡灌洗液中IgE呈负相关(n r=-0.55,n P<0.05)。T-B细胞共培养实验发现,n Sp-n a-/-小鼠脾脏来源Tfh细胞具有更强的辅助B细胞合成IgE抗体的能力(n t=3.18,n P<0.05)。后续的体外T细胞刺激实验证实,hSP-A处理可以上调培养体系中IFN-γn +Tfh细胞比例与IFN-γn +Tfh/IL-4n +Tfh的比值(n t值分别为6.34和3.16,n P值均0.05)。n 结论:异常的Tfh细胞亚群分布与哮喘的发生相关,SP-A可能通过稳定Tfh细胞亚群的分布,从而缓解哮喘气道炎症反应。“,”Objective:To explore whether surfactant protein A(SP-A) takes effect in the pathogenesis of asthma by modulating follicular helper T cells(Tfh).Methods:6~8 weeks old female wild type(WT) and SP-A gene knockout(n Sp-n a-/-) mouse on C57BL/6J background were immunized by OVA and aluminum hydroxide(WT asthma mouse: 8 cases, n Sp-n a-/- asthma mouse: 8 cases), and PBS control groups were established(WT control mouse: 6 cases, n Sp-n a-/- control mouse: 4 cases). HE staining was used to visualize the pathological changes in lung and flow cytometry was used to detect the distribution of Tfh cells and subsets in spleen and mediastinal lymph nodes.Tfh subgroups were defined as interferon-γ(IFN-γ)n + Tfh, interleukin(IL)-17n + Tfh and IL-4n + Tfh cells, according to their intracellular IFN-γ, IL-17 and IL-4 expression respectively.The level of immunoglobulin(Ig) E in bronchoalveolar lavage fluid(BALF) and T-B coculture system was measured by enzyme linked immunosorbent assay(ELISA). Besides, na?ve T cells were treated by human SP-A(hSP-A) in vitro, and the distribution of Tfh subsets was measured 5 d later.n Results:We have found that a large number of inflammatory cells infiltrated in BALF and the lung tissue of mouse after OVA challenged.The total number of inflammatory cells in BALF of WT and n Sp-n a-/- asthma mouse was significantly higher than that of the corresponding control mouse[(11.38±1.43)×10n 4vs(6.40±0.68)×10n 4, (14.38±1.52)×10n 4vs(6.25±0.48)×10n 4, n t values were 2.61 and 3.64, respectively, all n P values <0.05]. In additon, the level of total IgE in BALF of WT and n Sp-n a-/- asthma mouse were significantly higher than those of the corresponding control mouse[(16.85±2.50) pg/mL vs(4.94±2.01) pg/mL, (25.52±3.17) pg/mL vs(8.05±2.90) pg/mL, n t values were 3.63 and 3.58, respectively, all n P values <0.05)], and the level of IgE in n Sp-n a-/- asthma mouse increased significantly than that of WT asthmatic mouse(n t=2.20, n P0.05). But the subgroup composition of Tfh cells changed. The percentage of IL-4n + Tfh cells was positively correlated with the level of IgE(n r=0.50, n P<0.05), while the proportion of IFN-γn + Tfh cells was negatively correlated with the level of IgE(n r=-0.56, n P<0.05). The ratio of IFN-γn + Tfh/IL-4n + Tfh in the spleen in n Sp-n a-/- asthma mouse was significantly lower than that in WT mouse(n t=2.31, n P<0.05), and was negatively correlated with the level of IgE(n r=-0.55, n P<0.05). In the further study we have found that Tfh derived fromn Sp-n a-/- mouse have advantage in helping B cells synthesize IgE antibodies(n t=3.18, n P<0.05). Subsequently, vitro experiments showed that hSP-A treatment up-regulated the percentage of IFN-γn + Tfh cells and the ratio of IFN-γn + Tfh/IL-4n + Tfh(n t values were 6.34 and 3.16, respectively, all n P values 0.05).n Conclusion:The abnormal distribution of Tfh subsets is associated with the pathogenesis of asthma, and SP-A ameliorate asthma airway inflammation by stabilizing the distribution of Tfh cell subgroups.