人表皮生长因子基因转染原代角质形成细胞

Human Epidermal Growth Factor Gene Transfected Adult Epidermal Keratinocytes in vitro

目的：将带信号肽的人表皮生长网子基因转染原代成人角质肜成细胞，证实细胞活性及hEGF的有效表达，为后期的皮肤修复研究打下基础。方法：先酶切验证pcDNA3．1－hEGF，后消化成人皮肤组织，以Defined Keratinocyte—SFM（DKSFM）传代培养角质形成细胞并鉴定，脂质体转染质粒pcDNA3．1－hEGF人细胞，转基因细胞培养48h再作RT－PCR和Western—blot分析，上清分别进行放射免疫测定和MTT测定。结果：质粒peDNA3．1－hEGF上的hEGF序列经测序证实，双酶切后获得约230bp和5．4kb条带；成人角质形成细胞体外培养可快速稳定增殖，转hEGF基因细胞经RT—PCR扩增出一条约230bp的特异性条带。Westem—blot检测到hEGF表达明显升高；放射免疫法和MTT实验证实转基因细胞有稳定的hEGF蛋白分泌。结论：质粒pcDNA3．1－hEGF在脂质体介导下成功转染成人皮肤角质形成细胞，转基因细胞能分泌有生物活性的EGF；体外培养的成人皮肤角质形成细胞可见少量EGF分泌．

Objective： To transfect the human epidermal growth factor （hEGF） gene plasmid into adult epidermal keratinocytes, and to investigate the expression and secretiun of hEGF gene and the biological activities of hEGF protein in transfected keratinocytes. Methods： Primary adult epidermal keratinocytes isolated from human skin by two-step combined dissociation were cultured in defined keratinocyte-SFM （DKSFM）. The eukaiTotic expressiun plasmids pcDNA3. 1-hEGF were transteeted into keratinocytes in EGF-free medium mediated by Lipefectin 2000. After being cultured fur 48 hours, the transcription and expression of hEGF gene in transfected keratinocytes were investigated by RT-PCR and western-blot analysis. The biological activity of hEGF protein in supernatants secreted by transfected keratinoeytes was investigated by radioactive immunoassay and Hacat cells MTT assay. Results： Adult epidermal keratinocytes were all capable of being isolated and cultured in vitro steadily and rapidly. The plasmid pcDNA3. I-hEGF could be successfully introduced into cultured keratinocytes. These transgenic cells were able to secrete hEGF protein to certain extent, and have the biological activities to enhance the growth rate of Hacat cells in vitro. Conclusion： Adult epidermal keratinocytes in vitro transfected with exogenetic hEGF cDNA can express and secrete active hEGF.