| Ovotransferrin(OVT) is the main component of egg white protein, which has a variety of biological activities. In addition to the capacity of anti-bacterial, anti-inflammatory and antiviral, OVT is also involved in regulating the body’s immunity. Researches had showed that OVT can regulate T-cell proliferation in mouse. Dendritic cells(DCs) is the most powerful antigen-presenting cells in vivo, which specifically presented intracellular and extracellular antigen to naive T cells, triggering body immune response. Whether OVT has an effect on T cells by presenting antigen to DCs or affecting DCs downstream signaling, it has not defined. We successfully cultured murine bone marrow-derived dendritic cells in vitro, and examined the expression of the characteristic surface molecules of DCs, which stimulated by different concentration of OVT by flow cytometry. We measured the cytokine levels in DCs supernatant by ELISA, and MLRs measured the capacity of matured DCs inducing allogeneic mouse spleen T cell proliferation, Western-blot determinated the expression of MAPK signal pathway protein-p38 MAPK, JNK and ERK and their phosphorylation protein. By investigating the effect of OVT on DCs maturation and immune regulation, it can provide a theoretical basis for the subsequent tests. The results are as follows:(1) DCs stimulated by OVT on 6th day in vitro with different concentrations(10, 25, 50, 100, 200 μg/mL), OVT could significantly induced the expression of surface markers on DCs-CD80, CD40 and MHC-II molecules, which represented the maturity of DCs; DCs co-stimulated by different concentrations of OVT and LPS on 6th days in vitro, The expression of surface markers-CD80, CD40 and MHC-II molecules on DCs co-stimulated by 10~25 μg/m L(low concentration) OVT were significantly higher than that of the positive control group(LPS stimulated group), showing a synergistic effect on LPS, while 25~200 μg/mL(high concentration) OVT showed an antagonistic effect on LPS.(2) The proliferation of allogeneic mouse spleen T lymphocyte occurred when DCs matured by OVT stimulation. At the ratio of DCs: T cell =1:25, it induced the highest rate of T cell proliferation with different concentration of OVT. When DCs matured by co-stimulation, low concentration of OVT induced T cell proliferation significantly, while high concentrations of OVT can’t induce T cell proliferation. In the same concentration of OVT, the ratio of 1:25 led to the largest amount of T cells.(3) The content of TNF-α up-regulated when stimulated by OVT alone, and has a significant decrease at the concentration of 200 μg/m L; the expression of IL-10 presented continuously rise and reach the maximum at 200 μg/m L; the expression of IL-12p70 does not depend on the dose of OVT, it has no difference between the OVT dose groups and the control group.(4) The content of TNF-α showed a downward trend after rise firstly when DCs co-stimulated by LPS and OVT; the level of IL-10 increased as the concentration of OVT, and reached the maximum at 200 μg/mL; the production of IL-12p70 generated by costimulated DCs is significantly higher than OVT stimulated groups, but showed no significant difference among these costimulatory groups.(5) The signal pathway of DCs stimulated with OVT(25 μg/mL) for a period of time is ERK and JNK pathway-the downstream of MAPK signal path, and the expression of p-p38 MAPK up-regulated. The expression of JNK, ERK and their phosphorylation protein are different from control groups. DCs co-stimulated by LPS plus OVT(25 μg/m L) up-regulate the expression of p38 MAPK, JNK and their phosphorylated proteins, but the expression of ERK and its phosphorylation protein declined. |
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