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。
Abstract:
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.