The Experimental Study Of Effect On Immunocyte Treated With CpG-ODN And Lactobacilli-excreted RNA In Mice

Toll-like receptors (TLRs) are a important group of pattern recognition receptors(PRRs)expressed in different extent on the surface of lymphocyte, leukocyte and mononuclear phagocyte including macrophages and dendritic cells. TLRs can alternatively recognize pathogen associated molecular patterns (PAMPs) which are taken by pathogen and germ invading the body. CpG-oligodexynucleotides (CpG-ODN) is unmethylated nucleoside sequence from bacterium and virus genomic. As TLR 9 ligand, like LPS (TLR4 ligand) and other PAMPs, syntheic unmethylated CpG-ODN can imitate the function of bacterium DNA in vitro and has the function of activating innate immunity system cells. Besides, CpG-ODN also can act on tumor vaccine adjuvant to enhance vaccine activity. Whether Lactobacilli- excreted RNA have the effect on activating immunocyte or not and the involvement of TLR 9 in that process are to be defined.Objective:1. Effects of the proliferation on spleen cells, Macrophage and spleen-derived DC and the activity of neutral red phagocytosis in spleen–derived Macrophage treated with CpG-ODN and Lactobacilli- excreted RNA alone or combined with elemene combo-tumor cell vaccine.2. Effects on cytotoxicity against YAC-1 cells by DC and concentration of INF-γin the supernatant of DCs with different treatments.3. Effects of different treated BMDCs on the proliferation of spleen cells and the effect on expression of NF-κB on BMDC with different treatments.4. According to above experiments the functions of CpG-ODN and Lactobacilli-excreted RNA were compared and the similar functions of them were observed. And as tumor vaccine adjuvant their functions whether have similarity or not.Methods:1. The spleen cells were collected in vitro and on the basis of their growth feature DCs and Macrophages were isolated from spleen cells. Their proliferation was measured with MTT. The phagocytosis in Macrophages measured with experiment of neutral red phagocytosis.2. Bone marrow cells were cultured for 6 days in RPMI 1640 medium supplemented with GM-CSF and IL-4 to induce the bone-marrow derived DC. Their proliferation and MLR were measured with MTT. The expression of NF-κB on BMDC with different treatments was detected by Western Blot.3. DCs were treated with CpG-ODN and Lactobacilli-excreted RNA alone or combining with elemene combo-tumor cell vaccine in vitro for 24 hours. Then DCs and their cultural supernatant were collected. Their cytotoxicity against YAC-1 cells was evaluated with MTT assay; Concentration of INF-γin the supernatant of DCs was measured by ELISA.4. Morphological changes of DCs were observed in inverted microscope.Results:1. Effects of different treatment on the proliferation of immunocytes:①Spleen cells: The group of CpG-ODN, CpG-ODN+ Lysates and Lactobacilli-excreted RNA promoted the proliferation of spleen cells; and the group of Lysates and Lysates+ Lactobacilli-excreted RNA inhibited the proliferation of spleen cells.②Macrophage: The different treatments have no effect on the proliferation of Macrophage.③Spleen-derived DC: The group of CpG-ODN and Lactobacilli- excreted RNA promoted the proliferation of spleen-derived DC. The others inhibited the proliferation of spleen-derived DC.④Bone marrow-derived DC: The different treatments have inhibiting effect on the proliferation of BMDC.2. Effects of different treatments on the activity of neutral red phagocytosis in spleen–derived Macrophage: All groups could improve the neutral red phagocytosis in spleen–derived Macrophage, especially the group of CpG-ODN+ Lysates and CpG-ODN+ Lactobacilli- excreted RNA.3. Cytotoxicity against YAC-1 cells by DC with different treatments:①Cytotoxicity against YAC-1 cells by spleen-derived DC with different treatments: The cytotoxicity against YAC-1 cells by spleen- derived DC pulsed with CpG-ODN+ Lysates and CpG-ODN+ Lactobacilli- excreted RNA are significantly higher than the control group and the group of CpG-ODN+ Lysates is highest.②Cytotoxicity against YAC-1 cells by BMDC with different treatments: BMDC treated with different treatments could inhibit cytotoxicity .Except of the group of CpG-ODN+ Lysates; cytotoxicity of other groups is markedly lower than the control group.4. Concentration of INF-γin the supernatant of DCs with different treatments:①Concentration of INF-γin the supernatant of spleen-derived DCs with different treatments: Control group is 211.58±57.16 pg/ml; group of CpG-ODN is 456.00±31.68 pg/ml; group of Lysates is 300.22±53.91 pg/ml; group of CpG-ODN+ Lysates is 563.44±36.77 pg/ml; group of Lactobacilli- excreted RNA is 388.86±77.26 pg/ml; group of Lactobacilli-excreted RNA+ Lysates is 781.00±76.42pg/ml.The results show the remarkable difference, which is the different treatments enhance production of INF-γin spleen-derived DCs, exists between treatment groups and control group.②Concentration of INF-γin the supernatant of bone narrow-derived DCs with different treatments: Control group is 1777.77±57.16 pg/ml; group of CpG-ODN is 1096.00±48.41 pg/ml; group of Lysates is 1256.70±52.61 pg/ml; group of CpG-ODN+Lysates is 1043.17±41.25 pg/ml; group of Lactobacilli-excreted RNA is 1249.98±54.56pg/ml; group of Lactobacilli- excreted RNA+ Lysates is 1313.11±43.91 pg/ml. The results show the inhibition effect on production of INF-γin bone narrow-derived DCs with different treatments compared with control group.5. MLR: Effects of different treated BMDCs on the proliferation of spleen cells: All group can promote spleen cells proliferation, especially the group of CpG-ODN+ Lysates and CpG-ODN+ Lactobacilli-excreted RNA are higher than the other treated group.6. The expression of NF-κB on BMDC with different treatments: NF-κB up-regulate expression on BMDC.Conclusion:Our results show that CpG-ODN and Lactobacilli-excreted RNA promoted the proliferation of spleen cells and spleen-derived DCs, whereas they inhibited proliferation of BMDC. They have no effect on proliferation of Macrophage but they can markedly enhance phagocytosis and also can improve the activity of tumor Lysates in Macrophage. Spleen-derived DCs pulsed with them can facilitate cytotoxicity against YAC-1 cells and secrete more INF-γ; In contrast, in some condition the cytotoxicity against YAC-1 cells by BMDC could be suppressed by them and they can reduce to be secreted INF-γby BMDC compared with control group. But BMDC treated with different stimulus can promote the proliferation of spleen cells and up-regulate expression of NF-κB on BMDC. In a word, the functions of CpG-ODN and Lactobacilli-excreted RNA have similarity in some extent in their functions and as immune adjuvant.