摘要
目的应用过表达转录因子Foxp3的小鼠巨噬细胞RAW264.7细胞模型,探讨转录因子Foxp3对RAW264.7细胞表型及免疫抑制相关基因的影响,从而为移植排斥提供新的细胞治疗措施奠定基础。方法采用脂质体转染重组质粒pIRES2-EGFP/mFoxp3及空载质粒pIRES2-EGFP至小鼠巨噬细胞RAW264.7细胞系,经G418筛选后,免疫荧光显微镜及流式细胞术检测GFP表达、RT-PCR检测Foxp3表达以鉴定表达效果;采用RT-PCR检测转染重组质粒pIRES2-EGFP/mFoxp3、空载质粒pIRES2-EGFP以及未转染质粒组的RAW264.7细胞表面分子CTLA-4、GITR及GITRL的表达水平;采用RT-qPCR的方法检测各组RAW264.7细胞iNOS及Arg1的表达水平。结果成功建立稳定表达Foxp3的巨噬细胞RAW264.7细胞模型;与转染空载质粒pIRES2-EGFP和未转染质粒的RAW264.7细胞相比,转染重组质粒pIRES2-EGFP/mFoxp3的RAW264.7细胞的表面分子CTLA-4、GITR及GITRL的mRNA表达水平明显升高(P<0.01),免疫抑制相关基因iNOS及Arg1的mRNA表达水平也明显升高(P<0.01)。结论转录因子Foxp3能够使RAW264.7细胞表面分子及免疫抑制相关基因的表达水平明显升高,可能发挥抑制免疫应答的作用,从而在治疗移植排斥中发挥作用。
Objective To study the effects of transcription factor Foxp3 on the phenotype and immune suppression related genes in RAW264. 7 cell applying RAW264. 7 cell overexpressing Foxp3 to provide a new cell treatment for transplant rejection. Methods Murine macrophage RAW264.7 cells transfected with the recombinant plasmid pIRES2- EGFP/mFoxp3 or empty plasmid pIRES2-EGFP were screened by G418. Transfection efficiency was confirmed by im- munofluorescence microscopy observation and flow cytometry detection of GFP and RT-PCR detection of Foxp3. The mRNA expression of surface molecule CTLA-4, GITR and GITRL were detected by RT-PCR. The mRNA expression of iNOS and Argl were detected by RT-qPCR. Results RAW264.7 cell line overexpressing Foxp3 was established. The mRNA expression of CTLA-4,GITR and GITRL in RAW264.7 cells overexpressing Foxp3 were significantly in- creased compared with control groups（P〈0.01） ; the mRNA expression of iNOS and Argl in RAW264.7 cells overex- pressing Foxp3 gene were significantly increased compared with control groups（P〈0.01）. Conclusion Foxp3 induces the surface molecules and immune suppression related gene expression in RAW264.7 cell and may play a role in treat- ment of transplantation rejection through inhibiting the immune response.
出处
《中国实验诊断学》
2013年第12期2126-2130,共5页
Chinese Journal of Laboratory Diagnosis
