母亲特定的农村微生物环境对儿童哮喘保护性免疫调节机制的研究

Along with the urbanization process, the morbidity of asthma and allergic disease increases quickly recently. A lot of research found that exposures to rural specific microbial environment in early life (embryos and infant) can reduce the risk of allergic disease in childhood. But the immune mechanism for this allergy protection is not very clear. Our preliminary work has found that regulatory T cells (Tregs) in farming newborn were enhanced in Toll-like receptors（TLRs) pathway partly. Accordingly we assume that maternal exposure to rural specific microbial environment could affect neonatal T cell response through TLRs pathway, producing Tregs and Th1 immune response raise and Th2，Th17 and Th9 immune response decline, which reduce the risk of allergic disease in childhood. In our study, we will detect the environmental microbial levels. And then we will investigate the effect of maternal farm microbial exposure on the following contents: 1）gene expression of TLRs and CD14 and the amount of regulatory T cells (Tregs), Th1, Th2, Th17 and Th9 cells in newborn; 2) the pathway of TLRs-MyD88-NF-κB on Tregs in newborn; 3) the intervention of Tregs in the early differentiation of Th0 to Th1, Th2, Th17 and Th9 cells. With animal experiment, we will make pregnant mice exposed to certain simulated farm microbial environment, and then investigate the immune characteristics of microbial stimulation-TLRs-Tregs/Th axis in newborn mice; the rest offspring mice will be induced to asthma model, whose allergic and airway inflammation will be measured. Our study will prove that allergy protection of farm microbial exposures environment in early life due to TLRs-Tregs/Th axis partly, which will found theoretical basis for early prevention to children allergic diseases such as asthma.

随着城市化进程，哮喘等过敏性疾病的发病率逐年升高。研究发现，生命早期（胚胎期和婴幼儿期）暴露于农村特定微生物环境可降低儿童过敏性疾病的发病，但具体机制尚不清楚。本研究检测农村和非农村孕妇生活环境中微生物含量。并观察母亲特定的农村微生物环境对新生儿TLRs表达和调节性T细胞（Tregs）及Th细胞亚群数量的影响；对新生儿Tregs上TLRs-MyD88-NF-κB途径的影响；对新生儿在Th0向Th1，2，9，17细胞亚群的早期分化过程中Tregs所起的作用的影响。然后通过动物实验，将孕鼠暴露于特定微生物环境，观察新生小鼠在微生物刺激-TLRs-Tregs/Th轴的免疫特点；并将子鼠制作哮喘模型，观察母亲特定微生物环境对后代过敏及气道炎症反应的保护机制。从而明确生命早期暴露于特定微生物环境通过TLRs-Tregs/Th轴对哮喘等过敏性疾病的保护性免疫调节机制，为早期预防儿童哮喘奠定理论基础。

随着城市化进程，哮喘等过敏性疾病的发病率逐年升高。研究发现，生命早期（胚胎期和婴幼儿期）暴露于农村特定微生物环境可降低儿童过敏性疾病的发病，但具体机制尚不清楚。本研究检测农村和非农村孕妇生活环境中微生物含量。并观察母亲特定的农村微生物环境对新生儿TLRs表达和调节性T细胞（Tregs）及Th细胞亚群数量的影响；对新生儿Tregs上TLRs-MyD88-NF-κB途径的影响；对新生儿在Th0向Th1，2，17细胞亚群的早期分化过程中Tregs所起的作用的影响。然后通过动物实验，将孕鼠暴露于适当的高微生物负荷环境中，观察孕鼠-新生鼠的安全性,存活率等情况; 并将子鼠制作哮喘模型，观察母亲适当高微生物负荷对后代过敏及气道炎症反应的保护机制。结果发现: 1.母亲有农村因素暴露史的新生儿在PPG-TLR2及LPS-TLR4免疫途径上存在调节性T细胞数量、特异基因表达、特异性细胞因子分泌及抑制功能的一定程度的上调，尤其对Th2细胞极性的抑制作用更强，从而降低新生儿今后发展为过敏性疾病的可能性。2.母亲对后代过敏性疾病的预防性保护作用可能与其生活环境中内毒素含量较高及接触禽畜和宠物较多有关,通过影响新生儿的TLRs-MyD88-NF-kB-Tregs免疫途径发挥作用。3.本研究成功建立高微生物负荷（泛福舒预处理）母鼠-子鼠模型，母鼠-幼鼠安全性较好，未发现明显副反应，体重及新生鼠成活率均无明显变化，在子鼠哮喘模型中，肺部病理可见气道炎症，小气道周围可见散在嗜酸性粒细胞；支气管灌洗液及外周血中IgE,IL-4分泌增加，INF-r分泌降低，证明哮喘模型制作成功。4.与母鼠无处理-子鼠哮喘组比较，母鼠泛福舒预处理-子鼠哮喘模型中子鼠肺组织炎症反应减轻更明显，过敏反应减弱, 而调节T细胞数量上调，肺组织中TLR2，TLR4,MyD88蛋白表达有增加的趋势，NF-kB有下降趋势。以上结果证明母亲适当的高微生物负荷可以减轻后代过敏反应的强度, 这种保护机制可能是通过TLRs- MyD88-NF-kB途经上调调节性T细胞发挥作用，为我们寻求早期预防儿童哮喘等过敏性疾病的发生奠定理论基础。