M1 type macrophage exosome vaccine as well as preparation method and application thereof
A macrophage and exosome technology, applied in the field of tumor immunotherapy drugs, can solve the problems of ineffective activation of immune response, high immunogenicity, toxic and side effects, etc., and achieves alleviation of immunosuppressive phenomenon, simple preparation method, and reduced toxicity. Effects of side effects
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Embodiment 1
[0035] The following takes a widely used antigen model OVA as an example to introduce M1 in detail. OVA - Preparation method of Exos, such as figure 1 shown, including the following steps:
[0036] (1) Preparation of M1 macrophages carrying OVA
[0037] Female C57BL / 6 mice aged 6-10 weeks were selected and fasted for at least 8 h the day before sacrifice. After soaking and disinfecting with 75% alcohol, operate on the ultra-clean bench, and all items used must be clean and sterile. Fix the mouse to the foam board, lift the mouse abdominal fur with forceps, and cut it with scissors, do not cut the inner skin. Then lift the endothelium and cut a small opening, hold it with tweezers, pour cold DMEM complete medium into the abdominal cavity, blow and suck a few times in the abdominal cavity, and gently touch the abdomen of the mouse with your fingers to fully lavage, collect the peritoneal lavage fluid, repeat several times Centrifuge (1000 rpm, 5 min), resuspend with DMEM com...
Embodiment 2
[0042] (1) M1 OVA -Evaluation of Exos immune activation ability in vitro
[0043] There are a large number of TAMs in the tumor microenvironment, and affected by the tumor microenvironment, most of them show an immunosuppressive M2 phenotype, which is a major obstacle for immunotherapy. So we adopted M1-Exos polarized macrophages as the M1 phenotype. like Figure 5 As shown, the results of confocal laser show that M1-Exos and M1 OVA -Exos can increase the expression of CD80 and decrease the expression of CD206 on the surface of RAW264.7. This shows that M1-Exos and M1 OVA -Exos can efficiently polarize macrophages to the M1 phenotype. Image 6 The in vitro T cell activation ability of the material was analyzed by flow cytometry. T cell Jurkat (clone E6-1) with PBS, OVA, M0-Exos, M1-Exos and M1 OVA - After co-incubation with Exos, centrifuge PBS to resuspend the cells and label CD4+T cells and CD8+T cells with fluorescent antibodies, respectively. Detected by flow cytom...
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