The Impaction Of The Expression Of IDO In Dendritic Cells From Rats Spleen On The Proliferation Of T Lymphocyte

Objective: To study the isolation, purification, expansion and culture of dendritic cells derived from rat spleen in vitro, investigate the expression of IDO mRNA and protein in DC treated with different concentration of IFN-γand the effect of IDO on allogeneic T lymphocyte proliferation.Methods: Extracting of rat spleen, through collagenase and red blood cell lysing buffer digest and lyses to obtain the spleen cell suspension, density gradient centrifugation to obtain mononuclear cells and the method of one-step adherence to remove lymphocytes from mononuclear cells. The culture system including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) was used to induce and culture DC. The morphology of DC observed by microphotograph, and the phenotype of DC analyzed with flow cytometry; Separately with different concentrations of IFN-γ(0,100,300,500U/ml) induced DC, the relative quantification of IDO mRNA expression were detected by real-time PCR, and the expression of IDO protein were detected by Western blot and analyzed by Quantity one image analysis system. DC induced by different concentrations of IFN-γwas co-cultured with T lymphocyte, and the proliferations of T lymphocyte were observed by mixed lymphocyte reaction (MLR).Results: The spleen-derived DC from rat cultured at 10 days with typical dendritic morphology. The expression of OX62 of cultured DC is above 80%. The positive expression of CD80 and CD86 were more than 75% and 90%. The expressions of IDO mRNA and IDO protein were increased gradually with the increase of concentration of IFN-γ, the difference between the groups was significant(P<0.05). After induced by different concentrations of IFN-γ, the ability to stimulate T lymphocyte proliferation of DC were reduced obviously as compared with control group(P<0.05), and with the increased of concentration of IFN-γT lymphocyte proliferation rate decreased gradually, the difference between the groups was also significant(P<0.05).Conclusion: we obtained the spleen-derived DC from rat succefully by ultilizing the modified method of cultivating and adhereing. DC derived from rat spleen were successfully isolated and cultured in vitro. IFN-γcould induce the expression of IDO of DC in vitro. IDO enzyme could inhibit T lymphocyte proliferation of MLR cultures, and IDO activity in DC in an IFN-γdose-dependent manner.