腺相关病毒基因修饰后间充质干细胞来源的外泌体免疫调节功能的研究

OBJECTIVE: To verify the effects and mechanisms of natural MSC-exosome in treating acute GVHD in mice, explore and establish a method for targeted modification of MSC-exosome, and verify the functions of the modified MSC-exosome. METHODS: In different doses of MSC-exosome groups and MSC group, weight loss in acute GVHD mice was observed; then the proliferation levels of activated T cells were measured through T cell activation experiment in vitro and OVA antigen-specific T cell activation experiment in vivo. AAV2YF3 mutants carrying PD-L1 and PD-L1-ITGB1 were obtained after the construction of recombinant expression vectors and were then applied to infect human MSC to modify their exosome. The immunoregulatory functions of the modified MSC-exosome were measured with the abovementioned methods. RESULTS: ①Mouse MSC-exosome（300 µg × 3 times）and MSC（1 × 10(6) × 3 times）effectively alleviated the weight loss in acute GVHD mice. ②Compared with IL-2, 10, 25 and 50 µg human MSC-exosome inhibited the proliferation of activated T cells in vitro, respectively, 86.0％（IL-2）, 40.0％, 39.6％, and 42.8％; compared with PBS, 50, 100 and 200 µg mouse MSC-exosome inhibited the proliferation of antigen-specific activated OT-1 cells in vivo, respectively, 42.6％, 33.1％, 14.2％, and 10.6％. ③ After the infection of AAV2YF3 mutant carrying PD-L1 or PD-L1-ITGB1, the positive proportion of MSC-exosome exceeds 40％ and 60％, respectively. ④Compared with the natural state, MSC-exosome modified by PD-L1 or PD-L1-ITGB1 showed better proliferation inhibitory effect in vivo and increased the proportion of Treg cells in vitro. CONCLUSION: MSC-exosome exhibited similar immunomodulatory effects with MSC. MSC-exosome after PD-L1 and PD-L1-ITGB1-targeted modification effectively inhibited the proliferation of activated T cells and increased the proportion of Treg cells.