摘要
目的:建立稳定表达Δ42PD1的293T细胞系,并探索其对AKT,NF-κB和Erk1/2磷酸化的影响。方法:分别将含有人PD1和Δ42PD1基因的真核表达质粒稳定转染293T细胞,用流式细胞术筛选稳定细胞系,用RT-PCR和Western blot进一步鉴定目基因的表达。将稳定细胞系分别于PBMCs进行孵育,以激活Δ42PD1和PD1,用流式细胞术检测细胞内AKT,NF-κB和Erk1/2的磷酸化水平。结果:通过流式细胞术筛选出稳定表达PD1和Δ42PD1的293T细胞系,RT-PCR和Western blot确认了目的基因的表达。与PBMC混合孵育后,293T细胞内AKT的磷酸化水平被Δ42PD1或PD1显著抑制,而NF-κB和Erk1/2的磷酸化水平没有显著变化。结论:本研究发现Δ42PD1与PD1的功能类似,可能是一种重要的抑制性免疫调节受体。
Objective: To establish Δ42PD1-expressing stable 293T cell line and explore the effect of Δ42PD1 on phosphorylation of AKT, NF-κB and Erk1/2. Methods: The human PD1- and Δ42PD1-carrying eukaryotic expression plasmids were stably transfected into 293T cells respectively. Stable cell lines were screened using flow cytometry, and confirmed by RT-PCR and Western blot. The cell lines were co-cultured with PBMC to activate PD1 and Δ42PD1, and then analyzed the phosphorylation of AKT, NF-κB and Erk1/2 via flow cytometry. Results: We obtained PD1- and Δ42PD1-stably expressing 293T cell lines by flow cytometry. The expression of genes of interest was confirmed by RT-PCR and Western blot. After co-culture with PBMC, phosphorylation of AKT but not NF-κB or Erk1/2 in 293T cells was significantly inhibited by Δ42PD1 or PD1. Conclusion: The present study found that functionally similar to PD1, Δ42PD1 could be an important inhibitory immune-regula- tory receptor.
出处
《免疫学研究》
2016年第1期1-7,共7页
Immunology Studies
基金
国家自然科学基金(31500750)
广东省自然科学基金(2016A030313030)
深圳市科技创新基金(JCYJ20140411111718169,JCYJ20150402111430650)
深圳市医药卫生三名工程资助项目。
