学术
Smad3选择性调节TGF-β1促进大鼠骨髓间充质干细胞成骨分化的实验研究
王运涛[1] 郑启新[2] 吴小涛[1] 郭晓东[2] 茅祖斌[1] 陈辉[1] 赵梓汝[1]
[1]东南大学附属中大医院骨科,南京210009 [2]华中科技大学同济医学院附属协和医院骨科,武汉430022
摘 要:
目的探讨转化生长因子β1（TGF-β1）信号转导通路下游信号分子Smad2、Smad3在TGF-β1促进大鼠骨髓间充质干细胞（MSCs）成骨分化中的作用。方法采用RT-PCR、Western blot检测TGF-β1促MSCs成骨分化过程中Smad2、Smad3mRNA和蛋白表达的变化;脂质体介导稳定转染Smad3ΔC,间接免疫荧光试验鉴定;采用RT-PCR检测转染细胞中碱性磷酸酶（ALP）和核心结合因子（cbfa1）mRNA的表达,用对硝基苯磷酸盐（PNP）法检测细胞ALP活性,用茜素红染色法检测细胞矿化能力,观察Smad3ΔC对MSCs成骨分化的影响。结果在TGF-β1刺激后24h,MSCs中Smad3的mRNA和蛋白表达量明显减少（P〈0.01）,而整个刺激过程中,Smad2的mRNA和蛋白表达量无明显变化（P〉0.05）;稳定转染细胞中c-Myc抗原阳性表达;受TGF-β1刺激后,MSCs和V-MSCs（空载体转染组）中ALP、cbfa1mRNA的表达水平、矿化能力明显高于Smad3ΔC-MSCs;随TGF-β1刺激时间延长,Smad3ΔC-MSCs中ALP活性增加缓慢,仅于48h有明显增加（P〈0.05）,但与同时段MSCs和V-MSCs中ALP活性相比,差异有极显著性意义（P〈0.01）。结论TGF-β1促进MSCs成骨分化这一生物学效应的输出受Smad3特异性和选择性的调节。[著者文摘]
文章出处:
《华中科技大学学报：医学版》-2007年36卷5期,701 -625-629,701页
栏目信息:
分 类 号:
Smad3 Selectively Regulates TGF-β1-induced Osteoblastic Differentiation of Rat Bone Marrow-derived Mesenchymal Stem Cells
Wang Yuntao , Zheng Qixin, Wu Xiaotao et al （1 Department of Orthopedics, Zhongda Hospital, Southeast University, Nanjing 210009 ;2 Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022）
Abstract:
Objective To observe the role of Smad2 and Smad3 in transforming growth factor β1（TGF-β1） promoting osteoblastic differentiation in rat bone marrow-derived mesenchymal stem cells（MSCs）.Methods The expression of Smad2 and Smad3 in MSCs and the influences of extraneous TGF-β1 on them were examined by RT-PCR technique and Western blot assays.The MSCs were transfected with the complexes of pcDNA3.0-Myc-Smad3ΔC and lipofectamine reagent.Immunofluorescence technique was performed to evaluate the c-Myc expression.In order to observe the osteoblastic characteristic in stable expression MSCs,ALP mRNA and core binding factor α1（cbfa1） mRNA were detected by RT-PCR technique,and ALP activity and mineralization were examined by PNP method and alizarin red staining respectively.Results The expression levels of Smad3 mRNA and protein were decreased markedly 24 h after TGF-β1 treatment（P〈0.01）.The total amount of Smad2 remained unchanged before and after treatment（P〉0.05）.The c-Myc was detected in stable expression MSCs.The relative levels of ALP mRNA and cbfa1 mRNA in Smad3ΔC-MSCs were significantly lower than those in MSCs or V-MSCs,as well as mineralization.Although the ALP activity in Smad3ΔC-MSCs was increased slowly with prolongation of TGF-β1 treatment and markedly after 48 h（P〈0.05）,there was obvious difference with control groups（P〈0.01） at the same time point.ConclusionThe biological output of TGF-β1 which promoting the osteoblastic differentiation is specifically and selectively regulated by Smad3 in MSCs.[著者文摘]
Key words:
transforming growth factor β1;mesenchymal stem cells;Smad2;Smad3
基金资助:
江苏省自然科学基金资助项目（No.BK2007107）;东南大学新进博士科研启动基金资助项目（No.9290002359）
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