胰蛋白酶原16对哮喘气道肌成纤维细胞分化的影响

目的 研究慢性哮喘发展过程中气管壁中肌成纤维细胞的异常分化与胰蛋白酶原16(trypsinogen16,TG16)表达量之间的关系,进一步探索气道基底膜增厚的具体机制.方法 利用SDS-PAGE蛋白电泳分析比较OVA诱导的慢性哮喘小鼠与对照鼠肺总蛋白谱的差异,筛选出差异条带进行质谱分析.对差异条带中的TG16基因从小鼠肺组织中进行克隆和表达,转染体外培养的小鼠胚胎成纤维细胞同时给予转化生长因子-1(TGF-β1)刺激,RT-PCR分析过表达TG16对细胞机动蛋白α亚型(α-actin)的mRNA水平影响.给予TG16的RNA干扰,利用Western blotting检测TG16的下降对α-actin的表达水平的影响.结果 OVA慢性致敏小鼠的肺组织蛋白TG16含量明显增高;在TGF-β1刺激下,与对照组(α-actin/GAPDH=3.20±1.36)相比,3T3细胞外源表达TG16会明显抑制α-actin的表达(1.78±0.50);同样在TGF-β1刺激下,与转染对照寡核苷酸的3T3细胞(2.78±0.50)相比,应用RNAi干扰抑制TG16表达的同时α-actin的表达显著上调(3.60±0.44).结论 首次发现和证实TG16可以抑制α-actin在成纤维细胞中的表达,这可能是哮喘发展过程中机体本身的一种保护性反应.

Effect of trypsinogen 16 on phenotypic switch of airway fibroblasts into myofibroblasts

OBJECTIVE: To investigate the relation between transdifferentiation of the airway myofibroblasts and the expression level of (trypsinogen16, TG16) in vitro and explore the mechanism of airway basement membrane thickening. METHODS: The total lung proteins were extracted from normal and OVA-induced asthmatic mice and the protein expression profiles were analyzed with SDS-PAGE. The differentially expressed proteins were isolated for analysis with liquid chromatography-mass spectrometry. TG16 was cloned from mouse lung tissue and subcloned into the expression vector pcDNA3.0 to generate a pcDNA3-TG16 plasmid. The vectors were transfected into mouse embryonic fibroblast 3T3 cells and cultured in MEM in the presence of transforming growth factor-beta1 (TGF-beta1). The mRNA levels of alpha-actin and the housekeeping GAPDH gene were analyzed with RT-PCR. Using RNA interference, TG16 expression was suppressed and the resultant alpha-actin or GAPDH protein levels were analyzed using Western blotting. RESULTS: In the total lung proteins from OVA-induced mice, a 25 000 Da protein was significantly enhanced in comparison with the protein profiles of normal mice. The protein band was identified to represent the protein of TG16. With TGF-beta1 stimulation, transfection with the plasmid pcDNA3-TG16 significantly suppressed the mRNA expression of alpha-actin (alpha-actin/GAPDH=1.78-/+0.50) in 3T3 cells as compared with the expression in cells transfected with pcDNA3.0 (3.20-/+1.36); transfection of the cells with TG16 stealth RNAi oligonucleotide to decrease TG16 mRNA level upregulated the protein level of alpha-actin (3.60-/+0.44) as compared with the alpha-actin protein level in 3T3 cells transfected with control oligonucleotide (2.78-/+0.50). CONCLUSION: TG16 can inhibit the expression of alpha-actin in fibroblasts, which might be a protective mechanism in the progression of airway remodeling in asthma.