趋化素在特发性肺纤维化中的表达及其保护作用研究

Expression and protective effect of chemerin in idiopathic pulmonary fibrosis

目的探讨趋化素在特发性肺纤维化(IPF)中的表达及其作用。方法分别使用定量PCR与蛋白质免疫印迹法测定对照与IPF患者肺部组织中趋化素的mRNA及蛋白表达水平,通过酶联免疫吸附实验检测临床血清样本中趋化素的含量。构建肺纤维化体外模型,将分离得到的原代成纤维细胞分成对照组、TGF-β组、TGF-β+趋化素组和趋化素组,使用免疫荧光法检测各组成纤维细胞α-平滑肌蛋白(α-SMA)表达水平。构建肺纤维化体内模型,采用随机分组法将C57BL/6小鼠分成对照组、博来霉素组、博来霉素+趋化素组和趋化素组,通过肺组织病理学染色观察各组小鼠肺纤维化严重程度。通过定量PCR与免疫组化染色法分别检测肺纤维化体外与体内模型中上皮间充质转化(EMT)标记基因的表达情况。结果与对照组相比,IPF患者肺组织与血清中趋化素表达量均下调。免疫荧光结果显示,TGF-β组α-SMA表达水平较对照组明显增强,TGF-β+趋化素组中α-SMA表达水平则与对照组相仿。病理学染色结果显示,博来霉素组小鼠较对照组肺组织纤维化病变明显,趋化素表达量显著下调;博来霉素+趋化素组小鼠肺组织受损程度则有所缓解。定量PCR与免疫组化结果显示,趋化素在A549细胞与小鼠肺组织中分别减弱了由TGF-β与博来霉素诱导的EMT作用。结论趋化素在IPF患者中表达降低;趋化素可能通过调控EMT而在肺纤维化的发生中发挥保护作用。

Objective To explore the expression and the role of chemerin in idiopathic pulmonary fibrosis(IPF).MethodsQuantitative PCR and Western blotting were used to determine the mRNA and protein levels of chemerin in lung tissues from IPF patients and the controls.Clinical serum level of chemerin was analyzed by enzyme-linked immunosorbent assay.The mouse lung fibroblasts isolated and culturedin vitro were divided into the control,TGF-β,TGF-β+chemerin and chemerin groups.Immunofluorescence staining was used to observe the expression ofα-smooth muscle actin(α-SMA).C57BL/6 mice were randomly divided into the control,bleomycin,bleomycin+chemerin,and chemerin groups.Masson and immunohistochemical staining were performed to evaluate the severity of pulmonary fibrosis.Expression of epithelial to mesenchymal transition(EMT)markers was detected by quantitative PCR and immunohistochemical staining in the in vitro and in vivo models of pulmonary fibrosis,respectively.ResultsCompared with the control group,the expression of chemerin was downregulated in both the lung tissue and the serum of IPF patients.Immunofluorescence showed that treatment of fibroblasts with TGF-βalone resulted in a robust expression ofα-SMA,whereas treatment with TGF-βand chemerin together exhibited the similar expression levels ofα-SMA as the control group.Masson staining indicated that the bleomycin-induced pulmonary fibrosis model was constructed successfully,while treatment of chemerin partially alleviated the damage of lung tissue.Immunohistochemical staining showed that the expression of chemerin in the lung tissue was significantly decreased in the bleomycin group.Quantitative PCR and immunohistochemistry showed that chemerin attenuated EMT induced by TGF-βand bleomycin both in vitro andin vivo.ConclusionsThe expression of chemerin was reduced in patients with IPF.Chemerin may play a protective role in the development of IPF by regulating EMT,providing a new idea for the clinical treatment of IPF.