摘要
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 investigate the stable expression and secretion of human interlukin 2 gene in transfected keratinocytes Methods Keratinocytes were transfected with lipofectamine and selected by G418. Then the samples were analyzed with the techniques of DNA dot blot,RNA dot blot,hybridization in situ,immunohistochemistry, Western blot and MTT. Results the positive signals were observed in transfected keratinocytes by DNA dot blot,RNA dot blot,hybridization in situ and immunohistochemistry. With Western blot analysis,a specific band exhibiting a molecular weight of 15 000 was detected in transfected keratinocytes,which was in acordance with that of IL 2 The expression of IL 2 can maintain for up to 1 month The amounts of IL 2 in the supernatants of two and four passages transfected keratinocytes were 27 7 U/ml and 15.0 U/ml, respectively. Conclusion Keratinocytes have the potential for stable gene expression and secretion of active transgene products. Thus, it is possible to use keratinocytes as a target cell for gene transfection,gene expression and even gene therapy
出处
《中华医学杂志》
CAS
CSCD
北大核心
1999年第9期705-707,共3页
National Medical Journal of China
基金
国家自然科学基金!3 95 70 6 5 6
