阻断TGF-βTGF-βRI通路对ATDC5细胞增殖,分化及细胞外基质的影响.docVIP

阻断TGF-β/TGF-βRI通路对ATDC5细胞增殖、分化及细胞外基质的影响 王 权，戚华兵，王晓凤，朱 莹，陈 林 （400042 重庆，第三军医大学大坪医院野战外科研究所，创伤实验室，骨代谢与修复中心，创伤、烧伤与复合伤国家重点实验室） [摘要] 目的 研究TGF-βRI阻断剂SB-505124对软骨细胞增殖、分化及细胞外基质的影响与机制。方法 利用Western blot检测不同浓度的SB-505124处理后，前软骨细胞系ATDC5中p-Smad2/3的变化。MTT法检测SB-505124处理对ATDC5细胞生长、增殖的影响及时间、浓度依赖性效应。Western blot检测c-Myc蛋白水平的变化。Real-time PCR检测SB-505124处理后软骨分化、细胞外基质合成及降解相关分子Collagen Ⅱ、Aggrecan及Adamts5表达与变化，并利用阿尔新蓝染色法检测SB-505124处理对软骨细胞系蛋白聚糖合成的影响。结果 Western blot检测示，SB-505124处理ATDC5细胞明显抑制TGF-β1激活的p-Smad2/3，并呈浓度依赖性；MTT检测示，SB-505124浓度和时间依赖性的抑制ATDC5细胞增殖；Real-time PCR检测示，SB-505124处理明显抑制ATDC5细胞中Collagen Ⅱ、Aggrecan的表达，同时上调了Adamts5的表达；阿尔新蓝染色则提示SB-505124处理明显抑制ATDC5细胞中蛋白聚糖的合成。结论 利用SB-505124阻断内源性TGF-β信号，可抑制软骨细胞增殖、分化及细胞外基质的合成，同时促进细胞外基质的降解。 [关键词] TGF-βRI；SB-505124；ATDC5；增殖；细胞外基质 [中图法分类号] [文献标志码] A [基金项目] 国家自然科学基金重点项目 [通信作者] 陈 林，E-mail：linchen70@163.com Effect of inhibiting TGF-β/TGF-βRI signaling on proliferation, differentiation and extracellularmatrix of ATDC5 cell Wang Quan, Qi Huabing, Wang Xiaofeng, Zhu Ying, Chen Lin（StateKey LaboratoryofTrauma, Burns and Combined Injury, Center of Bone Metabolism and Repair, Institute of Surgery Reserch, Daping Hospital, Third Militray Medical University, Chongqing, 400042, China） [Abstract] Objective To investigate the effect of TGF-βRI inhibitor SB-505124 on proliferation, differentiation and extracellular matrix of chondrocytes. Methods The expression of p-Smad2/3 in ATDC5 cells which were treated with different concentration of SB-505124 was analyzed by western blot. The effect of SB-505124 on proliferation of ATDC5 and its relationship with concentration was detected by MTT assay.The effect of SB-505124 on the expression of c-Myc protein was analyzed by western blot.The effects of SB-505124 on the marks of differentiation and extracellular matrix, including Collagen Ⅱ, Aggrecan and Adamts5, were analysed by Real-time PCR.The effect of SB-505124 on expression of sulfated proteoglycans was measured by Alcian blue stain