摘要
目的：观察外源性人碱性成纤维细胞生长因子（ｈｂＦＧＦ）基因导入正常角朊细胞后，能否表达及分泌ｈｂＦＧＦ，并探讨其机理；为开展临床创面基因治疗提供实验依据和奠定物质基础．方法：采用脂质体包埋ｈｂＦＧＦｃＤＮＡ的真核表达质粒ｐｃＤＮＡ３－ｈｂＦＧＦ转染培养的人角朊细胞，经Ｇ４１８筛选、克隆、扩大及传代培养含转基因的角朊细胞．用特异性ｎｅｏ探针原位杂交显示转基因角朊细胞内含有拟转入的ｐｃＤＮＡ３－ｈｂＦＧＦ质粒载体；抗ｈｂＦＧＦ抗体免疫细胞化学法观察转基因角朊细胞内ｈｂＦＧＦ的翻译表达；选择人真皮成纤维细胞为效应细胞，应用抗体中和法分析转基因角朊细胞分泌ｈｂＦＧＦ活性物质所引起的成纤维细胞增殖效应，了解ｈｂＦＧＦ向胞外分泌情况．结果：ｐｃＤＮＡ３－ｈｂＦＧＦ可经脂质体介导有效地转入靶细胞角朊细胞内；用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ｐｃＤＮＡ３－ｈｂＦＧＦ，胞浆内有阳性ｈｂＦＧＦ表达，并向胞外分泌ｈｂＦＧＦ活性物质，刺激真皮成纤维细胞增殖．结论：含外源性ｈｂＦＧＦ基因的角朊细胞可于体外纯化及扩大培养。
AIM: To investigate whether human keratinocytes modified with the cDNA of hbFGF could be used to produce and secrete hbFGF. METHODS: After transfected with the eukaryotic expression plasmid pcDNA 3 bhFGF using Lipofectame, the gene modified keratinocytes, purified and cloned with G418 (geneticin), were cultivated and passaged in vitro . The pcDNA 3 bhFGFs were identified in the gene modified keratinocytes by in situ hybridization with neo probe, which contains a special sequence of neo gene cloned in the pcDNA 3 hEGF. The immunocytochemistry was employed to display the hbFGF expression in the cells. The hbFGF activity was measured in the supernatant from medium used in pcDNA 3 hbFGF containing keratinocytes culture. The proliferation rates of the fibroblasts were compared, after the supernatant was added alone to human dermal fibroblasts or together with the neutralization of hbEGF antibody. RESULTS: The pcDNA 3 bhFGFs were transfected efficiently into the human keratinocytes with Lipofectame. After purified with G418, the pcDNA 3 bhFGF containing keratinocytes could be propagated and passaged in vitro , and expressed the hbFGF observably in the cytoplasm. The active hbFGF secretion from the keratinocytes demonstrated by the enhanced proliferation of the human dermal fibroblasts. CONCLUSION: In vitro purified and passaged exogenetic hbFGF cDNA modified human keratinocytes could express and secrete the active hbFGF from the cells.
出处
《第四军医大学学报》
1999年第5期404-408,共5页
Journal of the Fourth Military Medical University
基金
国家自然科学基金
