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  

Effect of blocking TGF-β/TGF-βRI signaling pathway on proliferation,differentiation and extracellular matrix of ATDC5 cells

作　　者：王权[1] 戚华兵[1] 王晓凤[1] 朱莹[1] 陈林[1] 

机构地区：[1]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,重庆400042

出　　处：《第三军医大学学报》2013年第10期917-921,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金重点项目(81030036)~~

摘　　要：目的研究转化生长因子βⅠ型受体(transforming growth factor beta typeⅠreceptor,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CollagenⅡ、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ATDC5细胞中蛋白聚糖合成的影响。结果 Western blot检测结果示: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检测结果示:SB-505124浓度和时间依赖性的抑制ATDC5细胞增殖;Real-time PCR检测结果示:SB-505124处理明显抑制ATDC5细胞中CollagenⅡ、Aggrecan的表达,同时上调了Adamts5的表达;阿尔新蓝染色结果提示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。Objective To investigate the effect of transforming growth factor beta type Ⅰ receptor（TGF-βRⅠ） inhibitor SB-505124 on the proliferation, differentiation and extracellular matrix of chondrocytes. Methods The expression of p-Smad2/3 in ATDC5 cells that were treated with different concentrations of SB-505124 was analyzed by Western blotting. The effect of SB-505124 on proliferation of ATDC5 cells was detected by MTT assay. The effect of SB-505124 on the expression of c-Myc protein was analyzed by Western blotting. The effect of SB-505124 on the markers of cell differentiation and extracellular matrix, including collagen Ⅱ, aggrecan and ADAMTS5 were analyzed by real-time PCR. The effect of SB-505124 on expression of sulfated proteoglycans was measured by Alcian blue staining. Results Western blotting results showed that the treatment with SB-505124 induced a decline of phosphorylated Smad2/3 （p-Smad2/3）, including carboxyl termini and linker region stimulated by TGF-β1 in ATDC5 cells. MTT assay results demonstrated that SB-505124 significantly inhibited ATDC5 cell proliferation in a dose-and time-dependent manner, and the expression of c-Myc protein was also inhibited. Real-time PCR showed that collagen Ⅱ and aggrecan were inhibited by SB-505124, while ADAMTS5 was elevated by SB-505124. Alcian blue staining showed that the staining intensity was reduced in SB-505124 treated cells. Conclusion SB-505124 can block endogenous TGF-β signaling pathway to inhibit cell proliferation, cell differentiation and synthesis of extracellular matrix, and to promote the degradation of extracellular matrix.

关 键 词：转化生长因子βⅠ型受体 SB-505124 ATDC5 细胞增殖 细胞外基质 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]