哮喘小鼠气道平滑肌细胞磷酸二酯酶4D基因启动子甲基化的研究

摘要

目的 观察屋尘螨诱导哮喘模型小鼠气道平滑肌细胞中磷酸二酯酶4D(phosphodiestarase 4D,PDE4D)基因表达水平,及PDE4D基因启动子区域甲基化水平的改变.方法 12只C57BL/6小鼠随机分为对照组和模型组,每组6只.模型组用屋尘螨(house dust mite,HDM)致敏建立哮喘模型,对照组用磷酸盐缓冲液代替.检测小鼠肺功能变化,观察小鼠肺组织病理变化,进行肺泡灌洗并对灌洗液中免疫细胞分类、计数.实时定量聚合酶链式反应(real-time polymerase chain reaction,Real-time PCR)检测气道平滑肌细胞中PDE4D基因mRNA的表达量,重硫酸盐测序分析PDE4D基因启动子区域DNA甲基化水平.结果 与对照组小鼠相比,模型组小鼠气道平滑肌细胞中PDE4D基因mRNA的表达水平显著升高(P＜0.05),PDE4D启动子区域的甲基化位点的整体甲基化程度显著下降(P＜0.05).此外,部分甲基化修饰位点改变.结论 哮喘小鼠中PDE4D基因启动子区域的甲基化水平改变,可能通过调控PDE4D基因表达,参与哮喘的发生发展.%Objective To investigate the mRNA expression of phosphodiestarase 4 D (PDE4 D) and the methylation status of CpG sites at the 5'promoter region of PDE4 D in airway smooth muscle cells (ASMCs) isolated from house dust mite (HDM) -exposed mice.Methods C57 BL/6 mice were randomly divided into 2 groups (n=6). The control group was treated with PBS, while the model group received HDM exposure. The pulmonary function, historical changes of lung tissues, and cell count in bronchial lavage were recorded. Real-time PCR was performed to assess the level of PDE4 D mRNA expression in ASMCs. The methylation level of the 5'promoter region of PDE4 D gene was examined by methylation-specific PCR. Results Compared with the control group, the promoter region of PDE4 D gene was demethylated in tracheal ASMCs isolated from HDM-exposed mice (P＜0.05), meanwhile the mRNA expression of PDE4 D was significantly increased (P＜0.05). Conclusion Methylation of the promoter region of PDE4 D may play an important role in the development and progression of asthma through regulation the expression of PDE4 D.