论文部分内容阅读
目的 观察外源性人表皮细胞生长因子 ( h EGF)基因导入正常角朊细胞后 ,能否表达及分泌 h EGF;为深入探讨h EGF的作用机制及临床应用奠定物质基础 .方法 脂质体包埋 h EGF c DNA的真核表达质粒 pc DNA3- h EGF转染人角朊细胞 ,经 G418筛选、克隆、扩大及传代培养转基因的角朊细胞 .特异性 neo探针原位杂交显示转基因细胞内的质粒载体 ;h EGF抗体免疫细胞化学法观察转基因细胞内 h EGF的表达 ;采用放射免疫分析测定细胞向胞外分泌 h EGF的情况 .结果 pc DNA3- h EGF可经脂质体介导有效地转染角朊细胞 ;G418筛选纯化转基因后的角朊细胞内含转入的真核表达质粒 ,胞质内有阳性 h EGF表达 ;并向胞外分泌 h EGF.结论 由真核表达质粒 pc DNA3- h EGF转染人角朊细胞获得的含外源性 h EGF基因的角朊细胞具有表达分泌 h EGF的功能
Objective To observe whether exogenous human epidermal growth factor (h EGF) gene can express and secrete h EGF after it is introduced into normal keratinocytes, and lay a material foundation for further exploring the mechanism and clinical application of h EGF.Methods Liposomes Human eukaryotic expression plasmid pcDNA3-h EGF was transfected into human keratinocytes and screened, expanded, subcultured and cultured keratinocytes by G418.The specific neo probe in situ hybridization showed that the intracellular H EGF antibody was used to observe the expression of h EGF in the transgenic cells and the secretion of h EGF by the cells was detected by radioimmunoassay.Results pcDNA3-h EGF could be effectively mediated by liposomes The keratinocytes were transfected with G418, and the transfected keratinocytes were transfected with eukaryotic expression plasmids containing positive h EGF in the cytoplasm and secreting h EGF.Conclusion The eukaryotic expression plasmid pcDNA3-h Keratinocytes with exogenous h EGF gene obtained by EGF transfection of human keratinocytes have the function of expressing and secreting h EGF