建立NF-κB核转位的转基因细胞模型

Construction of nuclear factor-kappa B transgenic cell models

目的建立NF-κB(nuclear factor-kappa B,NF-κB)转基因细胞模型。方法 RT-PCR方法扩增p65基因片段;分子克隆技术构建哺乳动物细胞表达载体;脂质体法转染不同细胞;Western blot验证蛋白表达;利用荧光可视化技术观察p65核转位的情况。结果成功获得与绿色荧光蛋白(GFP)融合的载体;并建立p65转基因细胞模型;转基因组中p65蛋白表达量明显高于对照组,给予LPS刺激BV-2后能够促进p65向细胞核转位;而转染后的PC12细胞,p65的核定位现象则呈现不均一性。结论成功建立p65转基因细胞模型,针对不同细胞建立的模型,可用于相关化合物的筛选。

Aim To construct nuclear factor-kappa B（NF-κB） transgenic cell models.Methods p65 cDNA fragments obtained through RT-PCR methods were inserted into pEGFP-C1 plasmid.Different cell lines were transfected with the recombinant plasmid pEGFP-C1-p65 via LipofectimeTM 2000,then exposed to LPS,and analyzed with laser scanning confocal microscope fluorescence microscopy（Leica TCS SP2）.Results The recombinant pEGFP-C1-p65 was verified through sequencing which confirmed that the p65 cDNA fragments were inserted into the mock plasmid.Western blot results demonstrated that p65 protein level was increased in pEGFP-C1-p65 transgenic model than that in mock group.Conclusion The NF-κB transgenic cell models are constructed successfully.