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  

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

作　　者：朱国强[1] 李袁飞[2] 冯晋斌[1] 胡德华[1] 

机构地区：[1]解放军264医院烧伤整形科,太原030001 [2]山西医科大学第一附属医院肿瘤科

出　　处：《西南国防医药》2010年第11期1169-1172,共4页Medical Journal of National Defending Forces in Southwest China

基　　金：军队医药卫生青年基金资助项目(06Q018)

摘　　要：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-1cDNA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)48h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。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.

关 键 词：肺成纤维细胞 角化细胞生长因子-1 骨髓基质细胞 真核荧光表达载体 

分 类 号：R977[医药卫生—药品]