Abstract
AIMS: To construct and evaluate rats' tolerogenic dendritic cells (DC) through induction by NF-κB Decoy method. METHODS: GM-CSF and IL-4 were used to transform rats's monocytes into DC, and DC were stimulated with LPS, NF-κB Decoy ODN, and loaded with Bovine Type II Collagen. The following methods were employed to phenotype DC: 1) Observation of cell morphology; 2) Evaluation of cell viability using trypan blue staining; 3) Purity determination of DC through detection of specific markers OX-62; 4) Evaluation of mature state of DC via the determination of the expression of CD80 and CD86; 5) Determination of stimulation capability towards the proliferation of lymphocyte and the secretion of INF-r and IL-10. RESULTS: The activity of DC was more than 92%, and the expression of OX-62 was more than 70%. Most of DC exhibited the phenotype of CD80(+)/CD86(-). Compared with control group and LPS-stimulation group, the less mature adhered cells and hairlike DC were observed in NF-κB decoy group. Significant reduction (p < 0.05) was observed for the positive expression and extension of CD80 and CD86 in cell surface. After loaded with calf type II collagen, the low expression of CD80 and CD86 remains to be existed. The stimulation capability of DC towards lymphocyte in NF-κB decoy group was lower than that in control group (p<0.05) and LPS stimulation group (p < 0.05). CONCLUSION: NF-κB Decoy ODN method can be successfully applied for construct rats' tolerogenic dendritic cells (DC) with stable morphology and phenotype. The tolerogenic DC exhibited immature immune phenotype, and low capability to stimulate lymphocytes.