RNAi对成牙本质细胞表达TRAF6干涉作用

Interference effect of vector-based siRNA on TRAF6 gene of odontoblast cells

目的采用针对小鼠肿瘤坏死因子受体相关因子6(TRAF6)的siRNA真核表达载体,探讨其对小鼠成牙本质细胞样细胞MDPC-23中表达TRAF6的干涉作用。方法将构建成功的2个针对TRAF6基因的siRNA真核表达载体(pSUPER-T和pSUPER-2T),通过脂质体介导瞬时转染MDPC-23细胞,RT-RCR和Western blot法检测其对转染后细胞中TRAF6的mRNA和蛋白表达干涉效果。结果 pSUPER-T和pSUPER-2T转染MDPC-23细胞后,RT-RCR法显示2组TRAF6 mRNA表达下调、Western blot结果表明2组TRAF6蛋白表达水平降低,其中pSUPER-2T转染组抑制TRAF6表达作用较强。结论针对TRAF6的siRNA真核表达载体,在MDPC-23细胞中有效发挥了TRAF6表达干涉作用。

Objective To construct vectors expressing siRNA against tumor necrosis factor receptor associated factor 6（TRAF6） gene and to examine its interference effect on murine odontoblast-like cell line（MDPC-23）.Methods Two target gene segments were synthesized and cloned into pSUPER vector,respectively,to construct two recombinant eukaryotic expression vectors（pSUPER-T and pSUPER-2T）.The two recombinant vectors were identified by enzyme digestion analysis and DNA sequencing.Then MDPC-23 cells were transfected with pSUPER-T or pSUPER-2T and the interference effect was detected by reverse transcription（RT）-PCR and western blot.Results The results of RT-PCR and western blot indicated that both vectors could effectively down-regulate the transcription and expression of TRAF6 gene,and that pSUPER-2T had better interference effect than pSUPER-T.Conclusion The transcription and expression of TRAF6 gene in MDPC-23 cells were inhibited effectively by the constructed RNAi eukaryotic expression vectors,which will facilitate further studies of TRAF6 function and its application in the treatment of disease.