人角化细胞生长因子-1的克隆及其在骨髓基质细胞中的表达

目的 从人胚肺成纤维细胞中克隆人角化细胞生长因子-1（KGF-1）编码区序列,构建真核荧光表达载体.方法 从人胚肺成纤维细胞中提取总RNA,通过反转录 PCR获取KGF-1 cDNA编码区序列,将其与pIRES2-EGFP质粒载体连接,构建重组表达载体pIRES2-EGFP/KGF-1,测序鉴定.将pIRES2-EGFP/KGF-1瞬时转染骨髓基质细胞（marrow stroma cells,MSCs）48 h后,免疫荧光法检测骨髓基质细胞是否存在KGF-1蛋白的表达.结果 构建的重组质粒pIRES2-EGFP/KGF-1经序列分析示与国外文献报道一致.KGF-1免疫荧光法激光共聚焦检测结果证实,pIRES2-EGFP/KGF-1转染骨髓基质细胞后,存在KGF-1蛋白的表达,定位于细胞浆内.结论 成功构建重组真核荧光表达载体pIRES2-EGFP/KGF-1,初步探讨了该基因在骨髓基质细胞中的表达,为其在皮肤组织工程的运用奠定了基础.

Cloning of human keratinocyte growth factor-1 and its expression in marrow stroma cells

Objective To clone the encoding sequence of keratinocyte growth factor-1（KGF-1） from human embryonic fibroblasts for the construction of an eukaryotic fluorescent expression vector.Methods After total RNA was extracted from the fibroblasts in human embryo lung tissues,the KGF-1 sequence of cDNA was obtained by RT-PCR.Then the KGF-1 segments were inserted into pIRES2-EGFP vector and analyzed by restriction endonuclease mapping and DNA sequencing.The positive cloning of pIRES2-EGFP/KGF-1 was transiently transfected into marrow stroma cells （MSCs） with lipidosome.48 h later,the KGF-1 expression in the transfected MSCs was detected by immumofluorescence method.Results According to the sequence analysis,the recombinant plasmid pIRES2-EGFP/KGF-1 was consistent with the literature reported by Rubin.The immunofluorescenc and confocal laser scanning microscopy showed that the positive expression of KGF-1 protein could be clearly seen in the cytoplasm of MSCs after the recombinant plasmid transfected MSCs for 48 h.Conclusion Eukaryotic fluorescent expression vector of pIRES2-EGFP/KGF-1 is successfully constructed in this study.Meanwhile,the expression of KGF-1 gene has been preliminarily investigated,which can make a foundation for its use in the field of dermal tissue engineering.