摘要
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
AIM To investigate whether human keratinocytes modified with the cDNA of hEGF could produce and secrete hEGF. METHODS After being transfected with the eukaryotic expression plasmid pcDNA 3hEGF using Lipofectame, the gene modified keratinocytes, purified and cloned with G418 (geneticin), were cultivated and passaged in vitro. The pcDNA 3hEGFs were identified existing in the gene modified keratinocytes by in situ hybridization with neoprobe, which contained a special sequence of neogene cloned in the pcDNA 3hEGF. Immunocytochemistry was employed to show the hEGF expression in the cells. The hEGF level was measured in the supernatant from the medium used in the pcDNA 3hEGF containing keratinocytes culture by radioactive immunoassay. RESULTS The pcDNA 3hEGFs were transfected efficiently into the human keratinocytes with Lipofectame. After being purified with G418, the pcDNA 3hEGF containing keratinocytes could be propagated and passaged in vitro, and express the hEGF observably in the cytoplasm. The hEGF was detectable in the supernatant of the modified keratinocytes. CONCLUSION The human keratinocytes modified with exogenetic hEGF cDNA can express and secrete hEGF.
出处
《第四军医大学学报》
2000年第5期642-645,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金资助项目!(39600153)
