转化生长因子-β3融合蛋白促进骨髓基质干细胞向软骨分化的研究  

The promotion of chondrogenesis of marrow stromal stem cells by Transforming growth factor-β3 fusion protein in vitro

作　　者：郑东[1] 刘国辉[1] 杨述华[1] 许伟华[1] 冯勇[1] 李进[1] 叶树楠[1] 杨操[1] 傅德皓[1] 唐欣[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院骨科,武汉430022

出　　处：《中华实验外科杂志》2010年第1期104-107,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30471753）

摘　　要：目的 构建潜在相关肽（LAP）为潜伏位点,基质金属蛋白酶（MMP）切位点为导向位点,转化生长因子（TGF）-β3活性部分（mTGF-β3）为治疗位点的具有靶向治疗作用的新型TGF-β3融合蛋白LAP-MMP-mTGF-β3,并转染大鼠来源的骨髓基质干细胞（MSCs）,并检验其特异性的靶向治疗作用.方法 将人工合成的编码MMP酶切位点氨基酸的正反义DNA序列经基因重组定向克隆插入真核表达载体pIRES-EGFP中,获得pIRES-EGFP-MMP重组体,用逆转录-聚合酶链反应（RT-PCR）从大鼠胚胎组织中获取TGF-β3的LAP（781 bp）段和TGF-β3的活性片段mTGF-β3（313bp）,分别插入pIRES-EGFP-MMP中MMP的上游和下游,获得pIRES-EGFP-LAP-MMP-mTGF-β3重组质粒.然后将其转染入大鼠来源的MSCs,将转染后的MSCs在有MMP-1和无MMP-1的条件下进行球团培养,分别于第7、14、21天检测胶原2（COLⅡ）,Aggrecan（Agc）和TIPM的表达.结果 经过测序和酶切鉴定,构建pIRES-EGFP-LAP-MMP-mTGF-β3质粒;转染MSCs后,我们成功获得了LAP-MMP-mTGF-β3融合蛋白（39 kDa）的表达,并验证了其只有在MMP酶环境中才能被激活生成具有生物学效应的特异性的mTGF-β3二聚体（22.2 kDa）;球团培养中,MMP酶存在的条件下第21天COLⅡ、Agc、TIPM的相对表达量分别为1.45±0.07、1.61±0.09、1.80±0.08,无MMP酶存在的条件下第21天COLⅡ、Agc、TIPM的相对表达量分别为0.42±0.09、0.38±0.15、0.27±0.07.结论 构建了一种新型具有靶向治疗作用的融合蛋白LAP-MMP-mTGF-β3.Objective To construct a new TGF-β3 fusion protein （LAP-MMP-mTGF-β3） with targeted therapy function in which latency associated peptide,MMP enzyme and mTGF-β3 play the roles of latency,targeting and therapeutic effect,respectively.In addition,the specific targeted therapeutic effect was investigated by transfecting LAP-MMP-mTGF-β3 gene into rat marrow stromal stem cells （MSCs）.Methods The recombinant pIRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector pIRKS-EGFP.LAP and mTGF-β3 fragments were obtained from rat embryos by RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively,so as to construct the recombinant plasmid of pIRKS-EGFP-LAP-MMP-mTGF-β3,which was then transferred to rat MSCs.The genetically modified MSCs were cultured with medium in the presence of absence of MMP-1.Then the expression of Collagen Ⅱ,Aggrecan and TIPM was detected atthe clay 7,14 and 21.Results pIRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing analysis,and the mTGF-β3 fusion protein（39 kDa） was certified by Westen blot.When pIRKS-EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs,the medium with MMP enzyme could promote the chondrogenesis and the matrix production of genetically medified MSCs observably at the 21 st day （the relative expression of Coll Ⅱ,Agc,TIPM was 1.45±0.07,1.61±0.09,and 1.80±0.08 respectively）,and the medium without MMP enzyme could not promote the chondrogenesis and matrix production（the relative expression of Coll Ⅱ,Agc,and TIPM was 0.42±0.09,0.38±0.15,0.27±0.07 respectively）.Conclusion We got a new fusion protein LAP-MMP-mTGF-β3 by gene engineering.

关 键 词：软骨 分化 骨髓基质细胞 基因克隆 生长转化因子-β3 

分 类 号：R346[医药卫生—基础医学]