目的观察rhIL-33对人胚肺成纤维细胞增殖及其间充质细胞标记物的影响,探讨IL-33/ST2L-TRAF信号通路在肺纤维化形成过程中的作用。方法培养HELF细胞,PCR检测IL-33受体ST2L m RNA;不同浓度梯度的rh IL-33刺激细胞,MTT法检测不同时间点(24、48、72 h)IL-33对HF细胞增殖的影响;Real-time PCR方法检测IL-33刺激HELF后不同时间点(0、6、12、24、48、72 h)细胞标志性基因α-SMA m RNA、Vimentin m RNA、collagn I m RNA及TRAF-6 m RNA的变化;Western blot法检测细胞标志性蛋白α-平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)、I型胶原(collagen I)及关键性信号分子TRAF-6与下游信号分子ERK1/2、JNK、NF-kappa B等的改变。结果 IL-33能促进HELF的增殖,10 ng/ml的IL-33促增殖作用最强,且72 h最为明显;随着IL-33刺激细胞时间的逐渐延长,在0~72 h内α-SMA、Vimentin、collagen I在基因与蛋白水平均先上调后下调,信号分子TRAF-6、ERK1/2、JNK、NF-kappa B(P65)等亦呈现先上调后下调的趋势。结论 IL-33能促进HELF细胞增殖、活化及合成胶原,IL-33/ST2L-TRAF-6通路在此过程中起了关键作用,尤其是在病变早期炎症过程中作用最为明显。
To explore the role of IL-33/ST2L-TRAF-6 signaling pathway in pulmonary fibrosis, we investigated the effects of rhIL-33 on proliferations and expressions of mesenchymal components in the human embryonic lung fibroblasts (HELF). HELF cells were cultivated in vitro, and the expression of the receptor ST2L mRNA was analyzed by RT-PCR. Then, HELF were stimulated by rhIL-33 at different concentration for different times (24, 48, 72 h) respectively, and the proliferate rate of HELF was tested by MTT; changes of genes α-SMA mRNA, Vimentin mRNA, collagen I mRNA and the critical signal transducer TRAF-6 mRNA of IL-33 signal transduetion pathway were detected by real-time PCR at different time points (0, 6, 12, 24, 48, 72 h); Western blotting was employed to detect the protein expression of α-SMA, Vimentin, collagen I and critical signal transducer TRAF-6 as well as downstream molecules ERK1/2, JNK, and NF-kappa B (P65). Data showed that rhIL-33 promoted the proliferation of HELF (P 〈 0.05), especially at the concentration of 10 ng/ml for 72 h; Vimentin, collagen I increased and then reduced on mRNA and protein levels (P〈 0.05); signal transducers TRAF- 6, ERK1/2, JNK, NF-kappaB (P65) also increased and then reduced on protein levels (P〈 0.05). In conclusion, IL- 33 can promote fibroblasts to proliferate, activate and generate collagen, and IL-33/ST2L-TRAF-6 signal pathway playes critical role in this process, especially in earlier inflammatory reaction.