rhbFGF转染人皮肤成纤维细胞的研究

The Study on Transfection of rhbFGF into Human Skin Fibroblast

将重组人碱性成纤维细胞生长因子 (rhbFGF)基因克隆入真核表达载体pEGFP -N1质粒增强型绿色荧光蛋白 (EGFP)基因上游 ,并在其 5′端加上白介素 - 4的信号肽序列 ,形成融合基因 ,利用脂质体转染原代培养的成纤维细胞 ,荧光显微镜和蛋白质印迹杂交检测rhbFGF -EGFP融合蛋白的表达 ,细胞计数法观察bFGF对细胞生长的影响。结果表明成功构建了pEGFP -rhbFGF质粒 ,并经脂质体介导有效地转入原代培养的人皮肤成纤维细胞内。用G4 18筛选纯化转基因后的细胞能够向胞外分泌rhbFGF活性物质 ,明显促进成纤维细胞自身的增殖。

The rhbFGF gene was cloned into the eukaryotic expression vector pEGFP-N1, just before the enhanced green flurecent protein (EGFP) gene. At the 5′ end of the fused pEGFP-rhbFGF gene, the signal peptide sequence of IL-4 was introduced in order that the pEGFP-rhbFGF fusion protein could be secreted into the medium. Liposome was used in the transfection. The expression of the fused gene was detected through fluorescent microscope and Western blot, the influence of rhbFGF gene on cell growth was observed under microscope. Results showed that the pEGFP-rhbFGF plasmid was constructed successfully and transfected efficiently into the human dermal fibroblasts with Liposome. The transfected fibroblasts can secret active rhbFGF outside of the cells and effectively stimulate the cell growth. bFGF transfection can steadly promote the proliferation of human fibroblasts.