目的：探讨CRISPR/Cas9 基因编辑技术敲除食蟹猴T细胞PD-1 基因对T细胞增殖、表型及IFN-γ、IL-2 分泌的影响。方法: 设计靶向食蟹猴PD-1 基因的gRNA，构建并提取质粒，分离食蟹猴外周血单个核细胞（peripheral blood mononuclear cell,PBMC），加入质粒DNA，用Lonza 4D电转仪进行细胞转染，转染48 h 后用流式细胞术和荧光显微镜技术检测转染效率。提取细胞基因组DNA进行PCR扩增及T7E1 酶切鉴定。在人源性CD3 抗体和IL-2 刺激下诱导食蟹猴T细胞增殖并绘制细胞生长曲线，PI 染色流式细胞术检测T细胞的细胞周期及CD4、CD8 表达水平，ELISA检测IFN-γ、IL-2 的分泌水平。结果: 转染48 h 后，荧光显微镜下见实验组出现绿色荧光蛋白表达的细胞，其转染效率为（21.6±3.2）%；实验组细胞基因组DNA PCR产物经T7E1 酶切显示3 条带，出现目的分裂条带（243、197 bp）。与未转染组比较，（1）实验组T细胞增殖缓慢、集落形成时间延迟、细胞体积较小、折光性较弱；（2）实验组处于G0/G1 期的T 细胞数显著增多（P<0.05）、G2/M 期细胞数显著减少（P<0.05）；（3）实验组T 细胞IFN-γ、IL-2 的分泌水平显著升高（均P<0.05），但CD4、CD8 表达水平比较差异无统计学意义（均P>0.05）。结论: PD-1 基因敲除可使食蟹猴T细胞阻滞于G0/G1 期从而抑制其增殖，同时上调了IFN-γ、IL-2 的分泌水平。

Objective: To investigate the effects of CRISPR/Cas9 gene editing mediated PD-1 knockdown on the proliferation,phenotype, IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys. Methods: gRNA targeting PD-1 gene of Cynomolgus monkey was designed, and the corresponding plasmid was constructed and extracted. peripheral blood mononuclear cells (PBMCs) of Cynomolgus monkeys were isolated, and plasmid DNAs were added for transfection by using Lonza 4D electrorotometer. FACS analysis and fluorescence microscopy were used to detect transfection efficiency at 48 h after transfection. Genomic DNAof T cells was extracted for PCR amplification and T7E1 digestion identification. The proliferation of T cells was induced under the stimulation of human CD3 antibody and IL-2, and the cell growth curve was drawn. PI staining flow cytometry was used to detect cell cycle and the expression levels of CD4 and CD8, and ELISA was used to detect the secretion of IFN- γ and IL-2. Results: At 48 h after transfection, the cells with green fluorescent protein expression in experimental group were observed under fluorescence microscopy with a transfection efficiency of (21.6±3.2)%. T7E1 enzyme digestion results showed that the PCR product of genomic DNA of cells in experimental group showed 3 bands after digestion, including the target cleavage bands (243，197 bp). Compared with non-transfected cells, the cells in experimental group exhibited slow proliferation, delayed colony formation, with small volume and weak refraction; the number of T cells at G0/G1 phase of the experimental group was significantly increased (P<0.05), while the number of cells at G2/M phase was significantly reduced (P<0.05); and the secretion levels of IFN-γ and IL-2 in the cells of the experimental group increased significantly (both P<0.05). However, the difference in the expression levels of CD4 and CD8 was not statistically significant between the two groups (both P>0.05). Conclusion: PD-1 gene knockout can arrest T cells in Cynomolgus monkey at G0/G1 phase, thereby inhibiting its proliferation and increasing the secretion of IFN-γ and IL-2 in the meanwhile.

云南省科技计划重大项目资助（No. 2016FC007）；云南省细胞治疗技术转化医学重点实验室资助（No. 2015DG034）