| Background: The high incidence and the difficulty of cure make people to continue working on novel solution and treatment of cancer. The vaccines targeting a variety of cancers have brought new hope to millions of patients. Most of the vaccines have been testing on clinical trail, currently, it has been recognized that vaccines have great potential in the cancer treatment. The Nanog plays an important role in maintenance of pluripotency of stem cell and expresses in many types of tumors especially in cancer stem cells and germ-cell tumor; however, it has no effect on normal adult tissues. Tumors with intense Nanog expression were associated with further disease progression, a greater degree of metastasis and shorter cancer related survival. Testis embryonal carcinoma(EC), one kind of highly malignant germ-cell tumors, occurs primarily in young adults. EC cells, which are malignant stem cells of teratoma, exhibit a striking overexpression of stem-related protein Nanog. Thus, we hypothesized that it would be very powerful by inducing specifically Nanog-based immune response to treat EC tumors. TLRs are a family of integral membrane proteins which locate primarily in immune cells such as dendritic cells(DCs) and macrophages.The Toll-like receptors(TLRs) function significantly in terms of mediating natural and adaptive immunes. The TLR ligands have been widely investigated, especially the synthetic TLR7 agonist, the only small molecule that has been identified. Due to the feasibility of synthesis and alteration of this molecule, it has become a hotspot in immunology research. This research project based on Nanog protein antigen, that primes a novel vaccine formula in conjunction with TLR7 agonist, thus stimulate much stronger immune response toward tumors cells.Methods: In this study, prokaryotic expression has been employed to express Nanog protein, then conjugate TLR7 and Nanog protein through chemistry reaction and,use ultraviolet spectrum for identification and calculate.We use imiquimod as positive contrast. We set 4 groups:TLR7-Nanog, Nanog, TLR7, PBS(n=6). ELISA were used to determine the in vivo and in vitro biological activity, at the end, observed the tumour suppressor effect on mose tumor model.Result: The Nanog protein was successfully expressed in BL21(DE3). We successfully conjugated the TLR7 agonist to the protein in EDC/NHS conjugate system, and separated the conjugation of TLR7-protein from the excess TLR7 agonist through 10,000 MWCO Microcon filtration device.The TLR7-Nanog conjuage vaccine was confirmed that the TLR7 agonist:Nanog ratio is 3:1,detecting by ultraviolet spectrum.Immune stimulation experiment in vitro: Murine lymphocyte cells were separated from spleen and treated with PBS, TLR7,Nanog,TLR7-Nanog, conjugation. Trained lymphatic harvested after treated 72 h, and IL-12 and IFN-γ cytokines levels were measured by ELSIA kit. Three groups of TLR7, Nanog, and TLR7-Nanog was demonstrated the effective immune stimulation, the TLR7-Nanog was the top one among them.We isolate PBMC from bone marrow and culture in 37℃,0.5%CO2。We induce PBMC to DC with GM-CSF and IL-4.We also isolate single lymphocyte cell from spleen. DC and single lymphocyte was co-culture.IFN-γ secreted from lymphocyte cells was checked in the supernatants after 72 h. The additional 30μg/m L Nanog protein administrated after the first stimulation in 24 h, culture till day 3, then detect proliferation of Nanog- specific T-lymphocytes and specific CTL cytotoxicity effect on tumor cells. TLR7-Nanog groups compared with the PBS control group, the spleen index increased significantly(p<0.01); the level of IFN-γ secretion increased significantly(p<0.01); In CTL assay, in comparison of TLR7-Nanog with PBS control groups, were displayed outstanding difference(p<0.05). Survival rate on tumor models: In this study, we investigated the anti-tumor effects of TLR7-Nanog vaccine on mouse F9 cells homografted in BLAB/C mouse model meanwhile in Window chamber model. The results show that the TLR7-Nanog has powerful anti-tumor effects, compared with the control group, TLR7-Nanog group was liver longer(p<0.05). TLR7-Nanog group was liver longer than Nanog group(p<0.05). The results suggest that TLR7-Nanog vaccine not only play an important role in the immune system, but also can inhibit tumor angiogenesis through TLR7 agonist. Conclusion: Nanog itself has a certain immunogenicity, but after TLR7 coupled to Nanog may be further sensitized the immunogenicity, thus enhanced the immune response mediated by TLR7. TLR7-Nanog anti-tumor effects mostly attributes to stimulating the cytotoxic T cell activity, meanwhile inhibiting the formation of microblood vessels in the tumor site. It can be seen that improve the immunogenicity of the tumor protein is a comprehensive and effective ways to kill the tumor cells. The tumor vaccine targeting on Nanog had no effect on their normal adult cells. However, the exact mechanism by which stimulation of this TLR7 agonist conjugated Nanog complex promotes immune responses remains unclear, and should be further investigated. |
PDF Full Text Request