人白细胞介素2基因转染角朊细胞稳定表达的检测

目的 观察人白细胞介素２（ＩＬ－２）基因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Ｇ４１８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以均显著转染组有阳性信号,Ｗｅｓ

Detection of stable expression of human Interlukin-2 gene in transfected keratinocytes

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