S100A9对肺成纤维细胞的增殖及活性的影响

Effect of S100A9 on human lung fibroblast proliferation and activity

观察S100A9对人胚肺成纤维细胞(human embryo lung fibroblast,HLF)增殖、合成细胞因子和胶原的影响,并探讨S100A9活化细胞的可能机制。用CCK-8法检测不同浓度S100A9(0～1000 ng/ml)体外培养人肺成纤维细胞24 h、48 h或72h的细胞增殖反应。选择100 ng/ml S100A9刺激HLF细胞24 h后,采用Real-time PCR检测细胞表达IL-6、IL-8、IL-1β、高迁移率族蛋白B1(HMGB1)、Ⅰ型胶原和Ⅲ型胶原mRNA水平;用ELISA测定细胞上清液中上述细胞因子的蛋白水平;Western blot法检测细胞表达晚期糖基化终产物受体(RAGE)蛋白的变化。与对照组相比,5～500 ng/ml浓度范围内的S100A9对细胞均有明显的促增殖作用。S100A9可显著提高HLF细胞表达IL-6、IL-8、IL-1β的mRNA水平,且细胞培养上清液中上述炎症因子蛋白水平亦升高;S100A9可促进细胞合成Ⅲ型胶原mRNA水平升高,但对促纤维化因子TGF-β、CTGF、IL-4 mRNA的表达无影响。此外,S100A9可诱导成纤维细胞表达RAGE基因和蛋白水平增多。以上结果说明,S100A9能促进人肺成纤维细胞增殖以及合成炎症因子、胶原增多,且S100A9可能通过与细胞表面受体RAGE结合而活化成纤维细胞,表明S100A9可能在肺间质纤维化的发病机制中起着相对重要的作用。

This study aims to investigate the proliferation activity and expressions of cytokines and collagen in human fetal lung fibroblasts （HI.F） induced by S100A9. Human fetal lung fibroblasts were cultured in vitro, and exposed to S100A9 with different final concentrations（0 ng/mlN 1000 ng/ml）for 24h, 48h and 72 h respectively. The proliferation of cells were tested by CCK-8 assay. IL-6, IL-8,IL-113, HMGB1, collagen- I and collagen-Ill mRNA were detected after the treatment of 100 ng/ml S100A9 for 24 h by real-time PCR. The protein levels of these cytokines in the supernatant of the cultured HLF were deter- mined by ELISA. The expression of RAGE in HLF was detected by Western blot. Compared with the controls, S100A9 pro- moted the proliferation of HLF at the concentration ranging from 5 ng/ml through 500 ng/ml. Both the expression at mRNA and protein levels of IL-6, 11.-8, IL-113 were obviously elevated in HLF after being treated by S100A9. Meanwhile, S100A9 significantly increased collagen-Ill mRNA expression by HLF compared to controls. However, there was no significant changes in TGF-13, CTGF and IL-4 mRNA in HLF after S100A9 treatment. In addition, RAGE mRNA and protein expressions were obviously elevated in HLF exposed to $100A9 for 24 h. Thus, S100A9 could induce proliferation of HLF, and enhance produc- tion of pro-inflammatory eytokines and collagen by HLF. It may activate fibroblasts through binding to RGAE on the cells, which suggests that S100A9 may play an important role in the pathogenesis of pulmonary fibrosis and may be a promising target for the treatment of this disease.