中文摘要：

目的构建和鉴定含有标记基因绿色荧光蛋白（GFP）的人胰岛素样生长因子-1（hIGF-1）基因真核表达载体,为基因治疗关节软骨缺损奠定基础。方法利用基因重组技术将GFP cDNA连接至pcDNA3.1-IGF-1中,构建真核表达载体pcGI,然后限制性内切酶酶切鉴定。将pcGI转染成纤维细胞,经G418筛选,荧光显微镜、原位杂交和免疫细胞化学检测GFP和hIGF-1的表达,流式细胞仪检测细胞周期。结果酶切分析证明重组的pcGI质粒含有GFP cDNA和hIGF-1 cDNA碱基片段,方向及大小正确。转染后的成纤维细胞,荧光显微镜观察可激发出绿色荧光,原位杂交和免疫细胞化学检测有hIGF-1的表达。流式细胞仪分析显示转染后成纤维细胞S期细胞增加,G1期细胞减少（P〈0.01）。结论含有GFP和hIGF-1基因的真核表达载体pcGI其可以在成纤维细胞内获得稳定表达,并有效的促进成纤维细胞的增殖,这为进一步开展IGF-1基因治疗软骨损伤修复的研究奠定了基础。

英文摘要：

Objective To construct and identify a human insulin-like growth factor-1 （hIGF-1） eukaryotic expression vector pcGI containing green fluorescent protein （ GFP）, and to study the effect of pcGI transfection on NIH3T3 fibroblasts for repair of articular cartilage defects. Methods The GFP cDNA was inserted into pcDNA3.1-hIGF-1 to construct the eukaryotic expression vector pcGI by gene recombinant technique. Recombinant pcGI transfected into fibroblast were selected with G418. The stable expression of hIGF-1 in the fibroblasts was determined by in situ hybridiztion and immunocytochemical analysis, and GFP was confirmed by fluorescence microscope. The effect of hIGF-1 overexpression on NIH3T3 fibroblasts proliferation was observed by cell cycle. Results Enzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector correctly contained GFP and hIGF-1 cDNA, presenting correct direction and size. The fibroblasts transfected sent out green fluorescence obseved by fluorescence. The hIGF-1 expressions were observed by in situ hybridiztion and immunocytochemical analysis. Flow cytometry analysis revealed increased cells in S phage and decreased cells in G1 phage （P 〈 0. 01 ）. Conclusions The GFP and hlGF-1 eukaryotic expression vector containing GFP marker gene has been successfully constructed and can stably be expressed in fibroblast, promoting the fibroblasts proliferation, which play the basis for further research for repair of cartilage injury treated by IGF-1 gene.