RESUMO
【Objective】 To investigate the preventive effects of early apoptotic splenic mononuclear cells induced by extracorporeal photopheresis (ECP) on acute graft versus host disease (aGVHD) in mice and explore the underlying mechanisms. 【Methods】 1) Splenic mononuclear cells were extracted from C57BL/6 mice and treated with different concentrations of 8-MOP (50 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, 600 ng/mL). After treatment, irradiate the cells with 2 J/cm2 of ultraviolet light. Then, use the Annexin V-FITC/PI apoptosis detection kit to assess the early apoptosis rate of the cells and determine the optimal concentration of 8-MOP for the experiment.2) There were 35 SPF-grade female BALB/C mice (H-2Kd) aged 6-8 weeks. After whole-body irradiation with 8Gy X-rays, the mice were divided into five groups: sham irradiation group received intravenous infusion of 0.2 mL of normal saline, the syngeneic bone marrow transplantation group received intravenous infusion of 0.2 mL of BALB/C mouse bone marrow nucleated cell suspension (including a cell count of 1×107), the allogeneic bone marrow transplantation group received intravenous infusion of 0.2 mL of C57BL/6 mouse bone marrow nucleated cell suspension (including a cell count of 1×107), the aGVHD group received intravenous injection of a mixture of C57BL/6 mouse bone marrow nucleated cells (including a cell count of 1×107) and splenic mononuclear cells (including a cell count of 1×107) in 0.2 mL, the ECP prevention group received pre-transplant intravenous infusion of 0.2 mL of ECP-treated splenic mononuclear cells of C57BL/6 mice (including a cell count of 1×107 ) 48 hours before transplantation, and on the day of transplantation, intravenous injection of a mixture of C57BL/6 mouse bone marrow nucleated cells (including a cell count of 1×107) and splenic mononuclear cells (including a cell count of 1×107 ) in 0.2 mL.The preventive effects of ECP on aGVHD were observed, and the concentrations of IFN-γ, IL-2, TNF, IL-4 and IL-6 in mouse serum were measured using CBA. Th1 cell counts were determined by flow cytometry. 【Results】 Different concentrations of 8-MOP (50 ng/mL,100 ng/mL, 200 ng/mL, 300 ng/mL, 600 ng/mL) were used to treat mouse splenic mononuclear cells. The early apoptosis rates (%), observed after treatment were as follows: (14.18±0.865) vs (16.76±0.407) vs (18.83±0.404) vs (19.27±0.404) vs (14.5±0.529). The appropriate concentration of 8-MOP was determined to be 200 ng/mL.In vivo experiment, the results showed that the aGVHD group had decreased survival rate, reduced body weight, and increased clinical scores compared to the syngeneic and allogeneic bone marrow transplantation groups (P<0.01), and the chimerism of bone marrow cells in mice after transplantation was over 90%. ECP significantly improved the survival rate of mice after transplantation, reduced clinical scores (P<0.05), and decreased the concentrations of Th1 cell cytokines in serum (P<0.05) and the counts of Th1 cells in the spleen (P<0.05). 【Conclusion】 ECP-induced early apoptotic single nuclear cells from the spleen can prevent the occurrence of aGVHD by reducing the Th1 response in mouse.