环磷酰胺联合树突状细胞分泌的外泌体和PolyⅠ:C的抗肿瘤作用研究

Dendritic cells derived exosomes combined with cyclophosphamide and PolyⅠ:C in antitumor chemoimmunotherpy

目的观察树突状细胞(DC)分泌的外泌体(exosomes)与环磷酰胺(CTX)、PolyⅠ∶C联合应用的抗肿瘤效果。方法重组鼠粒细胞-单核细胞集落刺激因子诱导骨髓来源的造血干细胞分化为髓系树突状细胞,肿瘤细胞L1210的冻融抗原负载树突状细胞,以超速离心结合膜过滤的方法提取外泌体;用得到的外泌体在DC存在的情况下刺激T细胞,应用cck-8试剂盒测量T细胞的增殖;制备抗原特异性的细胞毒性T淋巴细胞(CTL),通过DIOC-18联PI的方法测定CTL对肿瘤的杀伤作用;L1210细胞接种小鼠,制备荷瘤小鼠模型,分为实验组(exosomes联合CTX和PolyⅠ∶C)和对照组(1.PBS组,2.CTX组,3.CTX+PolyⅠ∶C组4.exosomes组)作治疗,观察肿瘤的生长速度,小鼠的生存期。结果T细胞的增殖实验中,平均吸光度:实验组为0.90±0.22,对照组为0.53±0.13,差异具有统计学意义(P<0.05)。负载肿瘤抗原的exosomes刺激的CTL在体外能够特异性杀伤肿瘤细胞L1210,在效靶比分别为5∶1和10∶1时实验组的杀伤率分别为(21.19±3.99)%、(30.56±3.85)%,对照组的杀伤率分别为(17.54±4.48)%、(19.38±4.68)%,10∶1时实验组与对照组相比杀伤率差异具有统计学意义(P<0.05);与对照组相比,实验组荷瘤小鼠的肿瘤生长速度明显降低,小鼠的生存期延长。结论经过肿瘤抗原负载的exosomes刺激的T细胞体外能够杀伤肿瘤细胞;exosomes联合CTX和PolyⅠ∶C能显著抑制荷瘤小鼠体内肿瘤的生长,延长荷瘤小鼠的生存期。

Objective To study the antitumor effects of exosomes derived from dendritie cells with eyelophosphamide and Poly I： C. Methods DCs derived from bone marrow were loaded with frozen-thawed peptide antigen from L1210 tumor eells. Exosomes were then secreted from DCs. The exosomes were isolated by ultracentrifugation and ultrafiltration. To analyse the antitumor effects, the T cell proliferation and the cytotoxicity of exosomes stimulated antigen-specific T cells were measured, and animal mortality and tumor growth were monitored. Results Exosomes were potent for inducing antigen-spe- eifie T cell proliferation. The absorption value of T cell proliferation test was （0. 90 ± 0.22 } ,while T cells without exosomes stimulation was（0. 53 ±0. 13）. With effeetor target ratios of 10：1 and 5： 1, （30.56 ±3.85）% and （21.19 ±3.99）% L1210 cells were killed respectively when using T cells pulsed with exosomes, while the killing by T cells not pulsed with exosomes were （ 19. 38 ± 4. 68 ） % and （ 17.54 ± 4. 48 ） %. Compared with mice treated with PBS or CTX or CTX ＋ Poly I ： C, mice injected with exosomes lived longer and the tumor growth was slower. The miee treated with CTX ＋ Poly I： C ＋ exo- somes lived much longer and the tumor growth was slow. Conclusion Exosomes are potent in killing L1210. Exosomes ean prolong the survival time of tumor model. Exosomes together with CTX and Poly I： C are efficient in suppressing tumor growth and prolonging survival time.