摘要目的 研究乙酰半胱氨酸(NAC)抑制人肺成纤维细胞增殖和胶原合成的机制.方法 分离培养人肺成纤维细胞,并以肺泡上皮来源的细胞系A549作为对照.将细胞分为对照组(不加任何处理)、转化生长因子-β_1(TGF-β_1)组(TGF-β_1为5μg/L)、NAC组(NAC为20 mmol/L)和联合组(5μg/L的TGF-β_1刺激24 h后换用不同浓度NAC继续作用24 h).用不同浓度NAC处理后,四甲基偶氮唑蓝法检测细胞增殖,流式细胞仪检测细胞周期;采用TGF-β_1刺激后加用NAC刺激,逆转录-PCR法检测Ⅰ型前胶原mRNA表达的变化,Western blot法检测cyclin E、α-肌动蛋白及信号转导与转录激活因子-3(STAT-3)蛋白的表达.结果 5、10、20、40 mmol/L的NAC对人肺成纤维细胞生长均有明显抑制作用,呈剂量依赖关系,而20 mmol/L的NAC对A549细胞无明显作用.10、20、40 mml\L的NAC可使G_0/G_1期细胞比例明显升高,S期细胞比例明显降低.经TGF-β_1刺激后,成纤维细胞Ⅰ型前胶原mRNA表达明显增加,而NAC可抑制TGF-β_1诱导或未经诱导的Ⅰ型前胶原表达.TGF--β_1可诱导cyclin E和α-肌动蛋白的表达,其相对密度分别为0.98±0.09和1.56±0.23,20 mmol/L的NAC能明显抑制cyclin E的表达,其相对密度为0.52±0.04,但却小能抑制α-肌动蛋白的表达.TGF-β_1组和NAC组α-肌动蛋白的表达(相对密度分别为1.56 ±0.23和1.63±0.20)无明显差别.结论 NAC能够抑制人肺成纤维细胞cyclin E蛋白的表达,使细胞增殖阻滞于G_1期,从而抑制成纤维细胞发生增殖,抑制其胶原合成.对TGF-β_1刺激前后α-肌动蛋白的表达无明显抑制作用.

abstractsObjective To study the mechanisms of N-acetylcysteine (NAC) in the inhibition of proliferation and collagen synthesis of human pulmonary fibroblasts. Methods The human pulmonary fibroblasts (HFB) were primarily cultured in complete medium of DMKM/F12, with the cell line A549 derived from alveolar epithelia as the control. Different concentrations of NAC were administrated, with or without stimulation by TGF-β_1, for 24 h. The cell proliferations were tested by methylthiazolyltetrazolium ( MTT) and cell cycle detected with flow cytometer. The mRNA expression of type Ⅰ procollagen was tested with RT-PCR. Proteins of cyclin E,α-SMA and STAT-3 were detected with Western blotting. Results The proliferation of HFB was inhibited significantly by NAC at different concentrations (5, 10, 20 and 40 mmol/L). NAC had no effects on proliferation of A549 at a dose of 20 mmol/L The cell proportion in G_0/G_1 phase was increased by NAC at different concentrations (10, 20 and 40 mmol/L) , while the changes in S-phase ratio were decreased significantly. Procollagen type Ⅰ synthesis was increased by TGF-β_1, significantly. NAC showed inhibition on procollagen type Ⅰ synthesis before or after stimulation with TGF-β_1 Expression of protein cyclin E and α-SMA was significantly induced by TGF-β_1, the relative indensity being 0. 98 ±0. 09 and 1. 56 ±0. 23 respectively. Induction of cyclin E by TGF-β_1, was attenuated significantly by NAC 20 mmol/L (0. 52 ±0. 04). But α-SMA was not changed by NAC 20 mmol/L (1. 63 ± 0. 20). Stimulation with TGF-β_1, and NAC had no effects on expression of STAT-3. Conclusions Inhibition on proliferation of HFB by NAC may be through the attenuation of cyclin E. Differentiation of fibroblasts into myofibroblasts WaS inhibited by NAC through inhibition on α-SMA.NAC directly inhibited collagen synthesis.