GDF5基因真核表达载体的构建及其在恒河猴骨髓间充质干细胞中的表达

目的克隆人软骨组织生长分化因子5（GDF5）基因及构建GDF5基因真核表达载体，观察其在恒河猴骨髓间充质干细胞（MSCs）中的表达情况。方法采用反转录聚合酶链式反应（RT-PCR）从人胎儿软骨组织克隆hGDF5基因全长cDNA，插入pEGFP-C2载体，构建重组真核表达质粒pEGFP-C2-GDF5。重组质粒脂质体介导法转染MSCs细胞，荧光显微镜观察报告基因的表达，RT-PCR法检测目的基因表达。结果成功克隆人软骨组织GDF5基因和构建GDF5真核表达质粒pEGFP-C2-GDF5，克隆在载体上的基因长度为1505bp，包含全部cDNA编码序列1505bp，测序显示与Genbank上的序列一致。重组质粒转染恒河猴MSCs细胞得到表达，绿色荧光蛋白在转染24h后开始表达，72h达高峰，然后表达逐渐减弱。转染后72h可检测到GDF5mRNA表达。结论人GDF5基因在恒河猴MSCs细胞的成功表达为应用恒河猴模型开展基于细胞的基因疗法修复骨和软骨损伤研究奠定了必要基础。

Construction of Eukaryotic Expression Vector of Growth/Differentiation Factor-5 Gene and Its Expression in Rhesus Bone Marrow Mesenchymal Stem Cells

Objective To clone growth/differentiation factor-5 （GDF5） gene from human cartilage tissues, construct its eukaryotic expression vector and observe its expression in rhesus bone marrow mesenchymal stem cells （MSCs）. Methods The full-length cDNA of GDF5 gene was obtained by RT-PCR from the total RNA which was extracted from human fetal cartilage tissue, and was inserted into the pEGFP-C2 vector to construct a recombinant eukaryotic expression plasmid, pEGFP-C2-GDF5, which was transfected into MSCs by liposome-mediated method. The reporter gene expression was observed by fluorescence microscopy and the target gene expression was examined by RT-PCR. Results The GDF5 gene cloning and construction of eukaryotie expression plasmid pEGFP-C2-GDF5 were successful. The cloned gene was 1505 bp in length including full-length cDNA of GDF5. Sequence assay showed it was consistent with the reported GDF5 sequence in GenBank. Recombinant piasmia could be expressed in MSCs after transfection. GFP began to express at 24 h, reaching a peak at 72 h, and then the expression decreased gradually. GDF5 mRNA expression could be detected at 72 hours after transfection. Conclusions Successful GDF5gene expression in rhesus MSCs provides a basis for the filture studies on cell-based gene therapy for bone and cartilage damage repair using rherus.