DAPT对肺纤维化小鼠Th17细胞分化的影响*

Effect of DAPT on mouse model of pulmonary fibrosis*

摘要:

目的 研究γ-分泌酶抑制剂DAPT阻断NOTCH信号通路对博来霉素致肺纤维化动物模型Th17细胞分化的影响，探索肺纤维化的发病机制。方法 选取15只昆明鼠按随机数字表法分为对照组、模型组及DAPT组，每组5只。对照组气管注入生理盐水（1.25?ml/kg），其余两组注入博来霉素（5?mg/kg）复制肺纤维化模型。DAPT组于模型复制次日起用DAPT溶液灌胃[5?mg/（kg·d）]，隔天1次，直到处死。于第28天处死动物，取左肺组织行苏木精-伊红（HE）染色观察肺泡炎和肺纤维化程度。实时荧光定量聚合酶链反应（qRT-PCR）检测肺组织NOTCH 1、白细胞介素-17（IL-17）及α-平滑肌肌动蛋白（α-SMA）mRNA的表达水平。酶联免疫吸附试验（ELISA）检测外周血NOTCH 1、IL-17的含量。流式细胞术检测辅助T细胞17（Th17）的比例。结果 模型组肺泡炎及纤维化程度较重，DAPT组炎症程度较模型组减轻，纤维组织大量减少。qRT-PCR结果显示，模型组NOTCH1、IL-17和α-SMA mRNA的表达水平均较对照组升高（P?<0.05），NOTCH 1水平与IL-17呈正相关（P?<0.05）；DAPT组较模型组下降（P?<0.05）。ELISA结果显示DAPT组的外周血IL-17及NOTCH 1含量较模型组下降（P?<0.05）。流式细胞术结果显示模型组CD3+ CD4+ IL-17+细胞数量增加（P?< 0.05），DAPT组较模型组降低（P?<0.05）。结论 DAPT阻断NOTCH信号通路的激活，抑制Th17的分化，减轻肺纤维化程度。

Abstract:

Objective To explore effect of γ-secretase inhibitor on mouse model of bleomycin-induced pulmonary fibrosis. Methods 15 mice were randomly divided into 3 groups: control group, model group and DAPT group (n?=?5). Pulmonary fibrosis model was established by intratracheal instillation of bleomycin (5?mg/kg). Mice in control group was treated by intratracheal instillation of saline (1.25?ml/kg). Mice in DAPT group were administered with DAPT solution by gavage 5?mg/(kg?d) every two days for 28 days. Mice were sacrificed at 28th day and the pathological changes of the lung tissues were observed by HE staining. The levels of NOTCH1, IL-17 and α-SMA mRNA in lung tissues was tested by real-time quantitative qRT-PCR. The serum level of NOTCH1 and IL-17 was detected by ELISA. The proportion of splenic Th17 cells in each group was detected by flow cytometry. Results HE staining result showed the mice in model group experienced the most severe pulmonary fibrosis and alveolar inflammation compared with control group (P?