摘要
目的探讨双向凝胶电泳技术筛选及鉴定转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导的肺成纤维细胞向肌成纤维细胞转化的差异蛋白质。方法将大鼠原代肺成纤维细胞分为对照组及5 ng/ml TGF-β1诱导组(1、24h),以双向凝胶电泳筛选差异蛋白,分离并进行质谱鉴定。以Western blot法、免疫荧光染色、免疫组织染色对5 ng/ml TGF-β1诱导(6~48 h)肺成纤维细胞转化为肌成纤维细胞过程中差异蛋白[Rho GDP解离抑制因子α(Rho GDIα)]进行验证并对矽肺纤维化相关指标[α-平滑肌肌动蛋白(α-SMA)和I型前胶原(pro collagen-I)蛋白]进行检测。通过建立矽肺大鼠模型对肺组织中差异蛋白进行验证并对矽肺纤维化相关指标进行检测。结果经蛋白组学技术筛选出TGF-β1诱导的大鼠原代肺成纤维细胞的9个差异蛋白,与对照组相比,TGF-β1诱导组有8个蛋白点表达上调,1个蛋白点表达下调,其中Rho GDIα表达上调(P<0.05)。Western blot及免疫组化结果显示,与对照组相比,肺成纤维细胞Rho GDIα、α-SMA和pro collagen-I蛋白水平随TGF-β1诱导时间延长而增加(P<0.05),免疫荧光染色显示Rho GDIα和α-SMA蛋白共表达。动式染尘大鼠肺组织Rho GDIα蛋白表达随染尘时间延长而增加(P<0.05)。结论双向凝胶电泳技术对TGF-β1诱导后大鼠原代肺成纤维细胞表达的差异蛋白进行了筛选,Rho GDIα可能与肌成纤维细胞转化密切相关。
Objective To screen and indentify the differential proteins in the transformation from primary pulmonary fibroblasts to myofibroblast of rats induced by transforming growth factor-β1(TGF-β1) using two-dimensional gel electrophoresis.Methods The primary rat pulmonary fibroblasts were divided into control group(0 ng/ml,1 and 24 h) and TGF-β1 stimulation group(5 ng/ml,1 and 24 h),protein samples extracted from the groups were separated by two-dimensional gel electrophoresis and analyzed by mass spectrometry. The differential protein,such as Rho GDP dissociation inhibitorα (Rho GDIα) was verified,and the silicosis fibrosis-related indexes such as α-smooth muscle actin(α-SMA) and pro collagen-I were detected in the transformation from primary pulmonary fibroblasts to myofibroblast of rats exposed to 5 ng/ml TGF-β1 for 6-48 h,by Western blot,immunohistochemistry and immunofluorescence. The differential protein was verified and the silicosis fibrosis-related indexes were determined in lung tissue of rats model of silicosis also. Results Nine differential protein points were screened by proteomics technology;Compared with the control group,eight protein spots were up-regulated and one protein spots were down-regulated in TGF-β1 stimulation group;The expression of Rho GDIα was up-regulated statistically and significantly(P<0.05).In primary lung fibroblasts exposed to TGF-β1,the expressions of Rho GDIα,α-SMA and pro collagen-I protein,observed by Western blot and immunohistochemistry,were higher compared with the control,and increased with the prolongation of exposure time(P<0.05);Co-expression of Rho GDIα and α-SMA was observed by immunofluorescence also.The expression of Rho GDIα in lung tissue of rats dynamically exposed to SiO2 increased with the prolongation of exposure time(P<0.05). Conclusion Two dimensional gel electrophoresis is used to screen the differentially expressed proteins in primary lung fibroblasts induced by TGF-β1. Rho GDIα may be closely related to myofibroblast transformation.
作者
耿菲
陈莹莹
姚婧昕
李世峰
高学敏
魏中秋
徐洪
杨方
GENG Fei;CHEN Ying-ying;YAO Jing-xin;LI Shi-feng;GAO Xue-min;WEI Zhong-qiu;XU Hong;YANG Fang(College of Public Health,North China University of Science and Technology,Tangshan,Hebei 063000,China;不详)
出处
《环境与健康杂志》
CAS
北大核心
2020年第8期697-702,753,共7页
Journal of Environment and Health
基金
国家自然科学基金(81972988)
唐山市科技计划项目(18130216a)
河北省卫生计生委员会医学科学研究课题计划项目(20191107)
