负载乳腺癌抗原的脐血树突细胞外泌体抗肿瘤免疫作用

The antitumor immunologic effect of exosomes secreted by human umbilical cord blood dendritic cells loaded with breast cancer antigen

目的:探讨负载乳腺癌抗原的人脐带血树突状细胞(DCs)分泌的外泌体的抗肿瘤免疫效应。方法:提取新鲜脐血单个核细胞,体外诱导分化为DCs,提取乳腺癌细胞MCF-7肿瘤抗原,并冲击脐血DCs,加入LPS诱导其成熟,利用流式细胞仪检测表型;超速离心法收获负载抗原的脐血DC外泌体;MTT法检测外泌体诱导的T淋巴细胞增殖反应及其激活的CTL对肿瘤细胞的杀伤效应。结果:脐血DC表面标志CD34、CD80、CD86、CD11c阳性细胞数与新鲜脐血单个核细胞相比明显增加(P<0.05);Western-Blot结果显示,负载乳腺癌的脐血DC外泌体携带有MHC-Ⅱ类、CD40、CD80、CD86分子;成熟脐血DC组及负载乳腺癌抗原脐血DC外泌体对同种异体来源的T细胞均有明显促增殖作用;负载乳腺癌抗原的脐血DC及其外泌体激活的CTL均能对乳腺癌细胞MCF-7产生显著地杀伤作用(P<0.05)。且在效靶比100∶1时,外泌体的杀伤作用高于负载抗原的脐血DC(P<0.05)。结论:负载乳腺癌抗原脐血DC分泌大量外泌体,可促进同种异体T细胞增殖,并能诱导特异性抗乳腺癌的免疫效应。

Objective： To explore the antitumor immunologie effect of exosomes secreted by human umbilical cord blood dendritic cells （DCs） loaded with breast cancer antigen. Methods： The mononuelear cells were abstracted from fresh umbilical cord blood, and then they were induced to differentiate to DCs ; tumor antigen of breast cancer MCF - 7 cells were abstracted to shock umbilical cord blood DCs, LPS was added to induce maturity of DCs, flow cytometry was used to detect phenotype; supercentrilugation method was used to collect exo- somes of DC in umbilical cord blood loading tumor antigen; MTT method was used to detect proliferative reaction of T lymphoeytes induced by exosomes and lethal effect of activated CTL on breast cancer cells. Results： The numbers of CD34, CD80, CD86, and CDIlc positive DCs in umbilical cord blood were statistically significantly higher than those of fresh mononuclear cells （P 〈 0. 05 ） ; the results of Western - Blot showed that exosomes of umbilical cord blood DCs loading breast cancer carried MHC -Ⅱ , CIM0, CD80, and CI）86 cytokines; the exosomes of mature umbilical cord blood DCs and umbilical cord blood DCs loading breast cancer antigen had obvious promoting effects on allogenic T lymphoeytes; umbilical cord DCs loading breast cancer antigen and CTL activated by the exosomes had significantly lethal effects on breast cancer MCF - 7 cells （ P 〈 0. 05 ） ; when the ratio of effect cells to target cells was 100： 1, the lethal effect of exosomes was statistically significantly higher than that of umbilical cord blood DCs loading breast cancer antigen （P 〈 0.05 ） . Conclusion： Umbilical cord blood DCs loading breast cancer antigen secret a great deal of exosomes, which can promote proliferation of allogenic T lymphocytes and induce specific immunological effect of breast cancer.