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目的 观察人白细胞介素 2 (IL 2 )基因在皮肤角朊细胞中的稳定表达及分泌情况。方法 lipofectamine介导法转染 ,以G4 18筛选出阳性克隆并做扩大培养 ,用DNA斑点杂交、RNA斑点杂交、细胞铺片原位杂交、免疫组织化学、Western印迹杂交分析及噻唑蓝 (MTT)法检测外源基因在角朊细胞内的表达及分泌情况。结果 DNA斑点杂交、RNA斑点杂交、原位杂交和免疫组织化学染色均显示转染组有阳性信号 ;Western印迹杂交分析显示转染组细胞在相对分子质量为 15 0 0 0处出现一条特异性着色带 ,其位置与IL 2的相对分子质量一致 ,IL 2表达时间可持续在 1个月以上 ;MTT法检测结果显示 ,第二代、第四代转染细胞培养上清中IL 2的活性分别是 2 7 7U/ml和 15U/ml。结论 外源基因在角朊细胞内能够持续、稳定表达基因产物 ,并能分泌具有生物学活性的蛋白质。用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。
Objective To observe the stable expression and secretion of human interleukin 2 (IL 2) gene in keratinocytes of skin. Methods The positive clones were screened by lipofectamine. The positive clones were screened by G418 and expanded. The positive clones were identified by DNA dot blot, RNA dot blot, in situ hybridization, immunohistochemistry, Western blot and MTT assay. Method to detect exogenous gene expression and secretion in keratinocytes. Results Dot blot hybridization, RNA dot blot, in situ hybridization and immunohistochemical staining showed that there was a positive signal in the transfection group. Western blotting showed that a specific color appeared at the molecular weight of 1500 in the transfection group With the same relative molecular mass of IL 2, and the expression time of IL 2 could last more than 1 month. The results of MTT assay showed that the activity of IL 2 in the second and fourth generation of transfected cells were respectively Is 277 U / ml and 15 U / ml. Conclusion The exogenous gene can persistently and stably express the gene product in keratinocytes and can secrete the biologically active protein. It is feasible to use keratinocytes as target cells for gene transfection, gene expression and even gene therapy.