基于iTRAQ技术的TGF-β1诱导肺原代成纤维细胞差异蛋白筛选

Screening of TGF-β1 induced lung primary fibroblast differential proteins based on iTRAQ technique

目的:采用同位素标记相对和绝对定量(iTRAQ)技术筛选转化生长因子(TGF)-β1诱导的大鼠肺原代成纤维细胞的差异蛋白,并在矽肺大鼠模型中进行验证。方法:5 ng·ml^-1 TGF-β1诱导成纤维细胞24 h,采用iTRAQ联合液相色谱-串联质谱(LC-MS/MS)技术筛选出与对照组比较的差异蛋白,用Scaffold软件对蛋白质进行验证,采用PANTHER在线工具进行生物信息学分析。10只雄性Wistar大鼠,体重(160±10)g,分为对照组、矽肺模型组,每组5只。动式染尘[SiO2浓度(50±10)μg·m^-3]24周构建矽肺大鼠模型。VG染色观察肺组织形态,免疫组织化学染色法和蛋白质印迹法检测胶原(COL)Ⅴ、血管细胞黏附因子(VCAM)、极低密度脂蛋白受体(VLDLR)、多配体蛋白聚糖2(SDC2)的表达。结果:共鉴定出1 648个蛋白,TGF-β1诱导组有196个蛋白较对照组表达变化≥1.20倍。矽肺模型组大鼠肺组织可见胶原沉积,与对照组相比,COLⅤ、VLDLR、SDC2、VCAM蛋白表达增强,差异均具有统计学意义(P<0.05)。结论:基于iTRAQ技术筛选出与TGF-β1诱导的大鼠肺原代成纤维细胞有关的差异蛋白,其中COLⅤ、VCAM、VLDLR、SDC2可能参与了对矽肺纤维化的调控。

Objective: To screen the differential proteins on rats lung fibroblasts treated with or without transforming growth factor(TGF)-β1 by using a isobaric tags for relative and absolute quantitation(iTRAQ) profile and identify some differential proteins in rats exposed to silica. Methods: 5 ng·ml^-1 TGF-β1 was used to induce fibroblasts for 24 h, and the differentially expressed proteins were screened out by iTRAQ combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS) technology compared with the control group. The Scaffold software was used to verify the proteins and the bioinformatics analysis was performed with PANTHER online tool. Ten male Wistar rats with weight of(160±10) g were divided into control group and silicosis model group, 5 rats in each group. Silicotic rats were made by a HOPE MED esposure control apparatus[SiO2 concentration(50±10) g·m^-3] for 24 weeks. VG staining was used to observe lung tissue morphology. The positive expression of collagen alpha-1(Ⅴ)chain(COL Ⅴ), vascular cell adhesion protein 1(VCAM), very low-density lipoprotein receptor(VLDLR), and syndecan-2(SDC2) were observed and measured by IHC staining and Western blotting. Results: A total of 1 648 proteins differentially expressed in response to TGF-β1 treatment and 196 proteins were found to be expressed at ≥1.2 fold relative to control. The silicotic lesions could be observed by VG staing in rats exposed to silica. The positive expression of COL Ⅴ, VLDLR, and SDC2 were observed in silicotic lesions by IHC stainging. Compared with control group, the expression of COL Ⅴ, VCAM, VLDLR and SDC2 increased in silicotic group(P<0.05). Conclusion: The results suggest that iTRAQ profile can be emplyed for identifying differential protein, COL Ⅴ, VCAM, VLDLR and SDC2 may have some roles on silicosis.