人白细胞介素-2基因在转染角质形成细胞中的稳定表达

目的观察人白细胞介素-2(IL-2)基因在转基因角质形成细胞中的稳定表达、分泌情况以及角质形成细胞作为基因治疗靶细胞的可行性. 方法分离原代皮肤角质形成细胞,无血清培养基培养,Lipofectamine介导转染,G418筛选,DNA斑点杂交、RNA斑点杂交、原位杂交、免疫组织化学、Western blot分析及MTT法检测IL-2在细胞中的表达及分泌情况. 结果 DNA斑点杂交、RNA斑点杂交、原位杂交及免疫组织化学染色均显示转染细胞组有阳性信号;Western blot分析显示转染组在相对分子质量(Mr)15000处出现一条特异性染色带,其大小与IL-2一致;第二代、第四代转染细胞培养上清中IL-2的活性分别是27.7 U*mL-1和15 U*mL-1. 结论外源基因在角质形成细胞中能稳定表达基因产物,并能分泌具有生物学活性的蛋白质,以角朊细胞作靶细胞进行基因转染、基因表达、甚至基因治疗的设计是可行的.

Stable expression of human interlukin-2 gene in transfected keratinocytes

AIM To investigate the stable expression and secretion of human interlukin-2 (IL-2) gene in transfected keratinocytes and assess the possibility of keratinocytes as a target cell for gene therapy. METHODS Primary epidermal keratinocytes were isolated, cultured in serum-free medium, transfected by Lipofectamine and selected with antibiotic G418. The expression and activity of IL-2 were assessed with techniques of DNA dot blot, RNA dot blot, hybridization in situ, immunohistochemistry, Western blot and MTT colorimetric assay. RESULTS Positive signal was found in transfected keratinocytes by DNA dot blot, RNA dot blot, hybridization in situ and immunohistochemistry. Western blot analysis showed a specific band exhibiting a molecular weight of 15 000 which was in accordance with that of IL-2. The activity of IL-2 in the culture supernatants was 27.7 U*mL-1 in 2nd-passage cells and 15 U*mL-1 in 4th-passage cells. CONCLUSION Keratinocytes have the potential availablity for gene stable expression and secreting active transgene products; This observations support the feasibility of keratinocytes as a target cell for gene transfection, gene expression and even gene therapy.