Effects Of Methylene Blue On T Cell Immune Function Of Spleen Of Neonatal Mice

Plasma treated with methylene blue and light has been used for clinical transfusion for many years,and it is a kind of virus inactivated plasma product approved for clinical transfusion in our country.Immune-mediated reaction is the most common adverse reaction of plasma transfusion.The residual methylene blue in plasma treated with methylene blue and light is much lower than the dose of clinical adverse reactions.The existing literature shows that methylene blue has no significant effect on immune cells when the concentration of methylene blue is as low as the concentration of residual methylene blue in virus inactivated plasma.T lymphocytes are the most important cells in adaptive immune response.The immune function of newborns is not fully developed,and T lymphocytes are different from adults.The purpose of this study was to study the effects of different concentrations and doses of methylene blue on T cell immune function of 7-day-old mice,so as to provide a theoretical basis for the safety of neonatal transnfusion of plasma treated with methylene blue and light and using methylene blue as a clinical treatment.Chapter 1 Effects of different concentrations of methylene blue on immune function of neonatal murine splenic T cellsObjective:To investigate the effects of different concentrations of methylene blue on 7-day-old murine splenic T cell proliferation,CD3+CD4+/CD3+CD8+subsets,secretion of cytokines IFN-yand IL-10,so as to provide a certain theoretical basis for the safety of transfusion of plasma treated with methylene blue and light and using methylene blue as clinical treatment.Methods:Splenic mononuclear cells of 7-day-old BALB/c mice were cultured with methylene blue at different concentrations for 72 hours in the presence of anti-CD3ε antibody/CD28 antibody and IL-2.The proliferation of CFSE-CD3+T cells and CD3+CD4+/CD3+CD8+ ratio were detected by flow cytometry,and IFN-yand IL-10 were quantitatively detected by ELISA.Results and conclusion:When the concentration of methylene blue was≦0.63μmol/L,the proliferation of splenic T lymphocytes of 7-day-old mice and the secretion of IFN-yand IL-10 of splenic mononuclear cells of 7-day-old mice were not affected.At≧ 1.25μmol/L,the proliferation of splenic T lymphocytes of 7-day-old mice and the secretion of IFN-γ and IL-10 of splenic mononuclear cells of 7-day-old mice were inhibited in a concentration-dependent manner.When methylene blue concentration was≦0.31μmol/L,the CD3+CD4+/CD3+CD8+ ratio of splenic T lymphocytes of 7-day-old mice had no effect.At 0.63-1.25μmol/L,the ratio of CD3+CD4+/CD3+CD8+of splenic T lymphocytes of 7-day-old mice increased in a concentration-dependent manner.Chapter 2 Effects of different doses of methylene blue on splenic T cell differentiation in neonatal miceObjective:To investigate the effects of different doses of methylene blue on CD3+T cells/nuclear cells,CD3+CD4+/CD3+CD8+ ratio of spleen in 7-day-old mice,so as to further provide a certain theoretical basis for the safety of transfusion of plasma treated with methylene blue and light and using methylene blue as clinical treatment.Methods:Twenty-five 7-day-old BALB/c mouse were randomly divided into five groups:group A(untreated),group B(normal saline),group C(methylene blue/mouse body weight 10mg/kg),group D(methylene blue/mouse body weight 20mg/kg),group E(methylene blue/mouse body weight 40mg/kg).After intraperitoneal administration of 0.01mL normal saline or methylene blue solution/mouse body weight(g)for 72h,the ratio of CD3+T cells/nuclear cells and CD3+CD4+/CD3+CD8+in spleen was detected by flow cytometry.Results andconclusion:There was no difference in the ratio of splenic CD3+T cells/nuclear cells and CD3+CD4+/CD3+CD8+between untreated group and normal saline group.There was no difference in the ratio of splenic CD3+T cells/nuclear cells and CD3+CD4+/CD3+CD8+between the group of 10,20mg/kg methylene blue/body weight and the normal saline group.The CD3+CD4+/CD3+CD8+ratio in spleen was significantly different in the 40mg/kg methylene blue/body weight group compared with the normal saline group,but there was no difference in the ratio of CD3+T cell/nuclear cell.