目的 制备T细胞免疫球蛋白和黏蛋白结构域1(T cell immunoglobulin domain and mucin domain protein-1，TIM-1)-Fc融合蛋白并探讨TIM-1-Fc融合蛋白对卵白蛋白(ovabumin，OVA)诱导的哮喘小鼠的干预作用及潜在作用机制。方法 通过基因工程技术获得TIM-1-Fc融合蛋白。采用腹腔注射OVA氢氧化铝溶液致敏建立过敏性哮喘小鼠模型，随机分为对照组、哮喘组和TIM-1-Fc干预组。每次干预前20 min，使用40 μL卵白蛋白生理盐水溶液(OVA-NS)滴鼻，TIM-1-Fc融合蛋白干预包括TIM-1-Fc滴鼻组(每只小鼠每次给予1 μg/40μLTIM-1-Fc滴鼻)和TIM-1-Fc注射组(每只小鼠每次给予6μg/200μLTIM-1-Fc腹腔注射)，每天1次，连续7 d，对照组用生理盐水替代。采用苏木精-伊红(HE)染色观察肺组织病理变化;采用流式细胞术检测小鼠外周血中辅助性T细胞2(type 2 T helper cells，Th2)、辅助性T细胞17(type 17 T helper cells，Th17)和调节性T细胞(Treg)比例及相关细胞因子水平。结果 成功构建TIM-1-Fc融合蛋白，成功构建OVA诱导的过敏性哮喘小鼠模型。与哮喘组相比，TIM-1-Fc融合蛋白干预后显著减轻了哮喘小鼠气道炎性损伤和肺组织损伤;TIM-1-Fc融合蛋白干预能显著降低外周血中TIM-1+CD4+T细胞和TIM-1+Th17细胞比例，使TIM-1 Treg细胞增多，显著降低Th2、Th17细胞比例，提高Treg细胞比例，调节哮喘中Th1/Th2和Th17/Treg免疫失衡。结论 TIM-1-Fc融合蛋白改善OVA诱导的过敏性哮喘小鼠气道炎症和肺组织损伤，其作用机制可能与TIM-1-Fc融合蛋白对Th1/Th2和Th17/Treg的免疫调节有关。
TIM-1-Fc Fusion Protein Regulates Th1/Th2 and Th17/Treg Immune Imbalance in Mice with Asthma
Objective To prepare T cell immunoglobulin domain and mucin domain 1 protein-1(TIM-1)-Fc fusion protein,and to investigate the intervention effect of TIM-1-FC fusion protein on ovabumin(OVA)-induced asthma mice as well as its po-tential mechanism.Methods Through genetic engineering,pcDNA3.1(+)-TIM-1-Fc plasmid was constructed and stably transfected into CHO cells.Cell culture supernatant was collected and purified,and TIM-1-Fc fusion protein was ob-tained.Mouse model of allergic asthma was established by intraperitoneal injection of OVA aluminum hydroxide solution.The mice were randomly divided into control group,asthma group and TIM-1-Fc intervention group.The TIM-1-Fc intervention group included TIM-1-Fc nasal drop group and TIM-1-Fc injection group.20 min before each intervention,40 μL ovalbumin sa-line solution(OVA-NS)was used for nasal drops.Protein TIM-1-Fc nasal drops group(1 μg/40 μL TIM-1-Fc nasal drops was administrated in each mouse)and TIM-1-Fc injection group(6 μg/200 μL TIM-1-Fc was injected intraperitoneally in each mouse)were interfered with TIM-1-Fc fusion,once a day for 7 days.Normal saline was used as replacement in the control group.Hematoxylin-eosin staining(HE)was used to observe the pathological changes of lung tissue.Flow cytometry was used to detect the proportion of type 2 helper T cells(Th2),type 17 helper T cells(Th17),regulatory T cells(Treg)and the levels of related cytokines in peripheral blood of mice.Results TIM-1-Fc fusion protein and OVA-induced allergic asthma mouse model was successfully constructed.Compared with asthma group,TIM-1-Fc fusion protein could significantly reduce airway inflam-matory injury and lung tissue injury in asthmatic mice.TIM-1-Fc fusion protein intervention could significantly reduce the pro-portion of TIM-1 CD4+T cells and TIM-1+Th17 cells in peripheral blood,increase the number of TIM-1+Treg cells,signifi-cantly reduce the proportion of Th2 and Th17 cells,and increase the proportion of Treg cells.It could modulate Th1/Th2 and Th17/Treg immune imbalances in asthma.Conclusion TIM-1-Fc fusion protein improves airway inflammation and lung tissue injury in OVA-induced allergic asthma mice,and its mechanism may be related to the immune regulation of Th1/Th2 and Th17/Treg by TIM-1-Fc fusion protein.
万方数据
维普
