| Hyperthermia can enhance activities of immune effector cells and induce redistributio n of immune effector cells, and have an impact on expressions of cytokines, thus regul ating immune functions and inhibiting tumor growth and metastasis.Synthetic oligodeoxynucleotides containing unmethylated CG dinucleotides (CpG-O DN) can induce classified immune reactions and maturation and proliferation of a variet y of immune cells including B cells, T cells, NK cells, monocytes, macrophages and de ndritic cells. These cells secrete various cytokines and cell surface adhesion molecules,1 eading to inflammation and Th1 immune response.Dendritic cells (DCs) are currently known as the most powerful antigen-presenting c ells which can activate initial T cells in vivo, and the key factors to start specific anti-t umor immune responses. Studies have shown that mast cells have an important role i n dendritic cell maturation and activation. Recently mast cell degranulation agent Compo und48/80 has been increasingly brought into popular attention in tumor immunity. Ginse ng polysaccharides, a kind of herbal extracts, can activate T cell immune response serv ing as the immune adjuvant and significantly increase the antibody levels. In addition, s tudies have shown that ginseng polysaccharides exhibit a wide range of effective dose and a small amount of toxicity.OBJECTIVE:1.to evaluate and compare the effects on tumor treatment of CpG oligonucleotide sequ ences, Compound48/80 and ginseng polysaccharides as vaccine adjuvants in combination with magnetic hyperthermia.2.to explore mechanism of CpG oligonucleotides sequences serving as immune adjuvant V S.METHODS:1.Preparation of magnetic nanoparticles:The Fe3O4 magnetic nanoparticles(MNPs), synt hesized using chemical co-precipitation method, were then surface modified with 3-amino propyltriethoxysilane, with the result of magnetic nanoparticles called ATPS-MNP. Partic le sizes, morphology and other characterizations of MNPs were analyzed by transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FTIR), and therm ogravimetirc analysis (TGA) respectively.2.Animal experiments:Female C57BL/6 mice were inoculated with mouse melanoma B 16F10cells on the right hind limb to establish tumor models. Experimental animals we re divided into five groups:control group, hyperthermia group, hyperthermia + CpG grou p, hyperthermia + C48/80 group, hyperthermia + Ginseng polysaccharide group. After ane sthesiawith sodium pentobarbital, A certain dose of magnetic fluid or immune adjuvant i n situ injected to the tumor. Hyperthermia under alternating magnetic field was then con ducted. Tumor growth and mice survival were observed.3.Detection:mice heart, liver, spleen, lung, brain, and tumors tissues were HE and Pruss ian blue stained for pathological analysis; Expression of IL-1β, IL-6 and TNF-αin seru m, and expression of immunoglobulin G type IgG 1, IgG2a and IgG2b in serum were dete cted using relevant ELISA kits. Expression of Toll-like receptors TLR1-9 and interme diate protein Myd88 were detected by Real time RT-PCR.RESULTS:1.Magnetic nanoparticles:the mean sizes of the magnetic nanoparticles were about 10nm. Good heating properties were observed in the alternating magnetic field.2.Animal experiment:Compared with the control, the growth of melanoma was signif icantly inhibited in each hyperthermia treatment group. And the difference was statistica lly significant (p<0.05). Survival spans were extended compared with the control group. The difference was statistically significant only five days after the last time of hyperth ermia.3.ELISA:Compared with control group, expression of IL-1βand TNF-αwere increase d sifnificantly in hyperthermia group and the three combined treatment group, the expres sion of IL-6 was slightly decreased; compared with controls, IgGl, IgG2a and IgG2b e xpressions were significantly increased, The ratio of IgG1/IgG2a or IgG1/IgG2b was fo und to be>1.0.4.Real-time PCR:Compared with control group and hyperthermia alone group, the ex pressions of TLR2, TLR4 and TLR9 increased remarkably in CpG-ODN combination group and Compound48/80 combination group.CONCLUSION:1.tumor growth of mice bearing melanoma were inhibited significantly by local magnet ic hyperthermia with the dose of 50℃×15min and survival was extended. Howeve r, skin injuries were also observed.2.Compared to the hyperthermia group, the differences of tumor inhibition were signifi cant only five days after the last time of hyperthermia in the adjuvant combination tre atment groups, and long-term immune function were observed; the effect of CpG-OD N adjuvant is more obvious than the other two adjuvants. CpG-ODN adjuvant induced T hl-type immune response and Toll-like receptor pathway dependent. |
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