Zheng Dong, Dan Yang, Huang Peng, Xia Tian, Yang Shu-Hua, Xu Wei-Hua, Yang Cao, Liu Guo-Hui, Liu Xian-Zhe, Feng Yong
Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2013 Aug;33(4):536-542. doi: 10.1007/s11596-013-1155-2. Epub 2013 Aug 1.
A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs). The recombinant pIRES-EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFP. LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups: experimental group (MMP group), negative control group (no MMP) and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen (ColII) was detected by using Alcian blue staining and immunohistochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and sequencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differentiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.
构建了一种具有靶向治疗功能的新型转化生长因子β3（TGF-β3）融合蛋白，并通过将LAP-MMP-mTGF-β3基因转染至脂肪来源干细胞（ADSCs）来研究其诱导软骨生成的可行性和靶向特异性。通过将编码合成MMP酶切割位点氨基酸的正义和反义DNA插入真核表达载体pIRES-EGFP中，构建重组pIRES-EGFP-MMP。利用逆转录聚合酶链反应（RT-PCR）获得LAP和mTGF-β3片段，并分别插入pIRES-EGFP-MMP中MMP的上下游，构建pIRES-EGFP-LAP-MMP-mTGF-β3重组质粒，并将其转染至ADSCs。将ADSCs培养并分为三组：实验组（MMP组）、阴性对照组（无MMP）和未转染组。在显微镜下观察形态变化，并在培养后第7天、14天和21天利用阿尔辛蓝染色和免疫组织化学染色检测蛋白聚糖和II型胶原蛋白（ColII）的表达。通过酶切和测序分析方法正确构建了pIRES-EGFP-LAP-MMP-mTGF-β3重组质粒。转染后成功表达了mTGF-β3融合蛋白，并且在MMP存在的情况下产生了活性蛋白mTGF-β3，其显著促进了ADSCs向软骨细胞的分化以及软骨基质的表达。新型融合蛋白LAP-MMP-mTGF-β3可靶向诱导ADSCs向软骨细胞分化，这将为未来软骨损伤修复的靶向治疗开辟前景。
