T-exo 负载DC诱导抗肝癌细胞免疫效应研究

摘要

Objective:To investigate the cytotoxic effects of CTL cell induced by DCs loaded with exosomes derived from hepatoma Huh-7 cells(T-exo).Methods: Exosomes derived from hepatoma Huh-7 cells were isolated and purified by combination of ultrafiltration centrifugation and sucrose density gradient centrifugation.Morphology of exosomes was observed under transmission electron microscopy and the expression of CD 9,CD63,HSP70 and AFP was detected by Western blot.DCs were induced with peripheral blood monocytes isolated from healthy donors.Flow cytometry was used to analysis surface markers of the DCs loaded with T-exo.WST-1 light absorption measurement was adopted to evaluate the T cell proliferation ability.Annexin-V/PI Flow cytometry were respectively used to examined cytotoxicity against the tumor cells.Results:Exosomes isolated and extracted from culture supernatant of Huh-7 cells presented as circular or elliptical vesicle with bilayer membrane , unequal in size , and with diameter of 50 to 100 nm.Western blot showed that the T-exo expressed CD9,CD63,HSP70 and AFP molecules.DCs loaded with T-exo caused significantly higher T cell pro-liferation and cytotoxic effect against AFP positive Huh-7 cells as compare to gainst AFP negative SMMC 7721 cells and un-loaded control group ( P<0.05 ).Conclusion: T-exosome loaded-DC can promote proliferation and induce significant cytotoxic effect of CTL against Huh-7 cells.%目的：观察肝癌细胞来源的外泌体（T-exo）负载DC体外诱导细胞毒T细胞（CTL）对肝癌细胞的杀伤作用。方法：采用超滤离心技术联合蔗糖密度梯度超速离心的方法从肝癌Huh-7细胞培养上清液中分离外泌体（ T-exo ），透射电镜鉴定形态，Western blot 检测外泌体相关蛋白CD9、CD63、HSP70及肿瘤抗原分子AFP的表达；分离健康供者外周血单个核细胞（PBMC）培养DC，流式细胞术鉴定负载T-exo 的DC细胞表型；用2-（4-碘苯）-3-（4硝基苯）-5-（2，4-磺苯基四氮唑）-2H-四唑单钠盐-1（ WST-1）法检测负载T-exo 的DC 刺激T淋巴细胞增殖情况；流式细胞术Annexin-V／PI双染法检测负载T-exo 的DC诱导CTL分别对AFP阳性Huh-7细胞及AFP阴性的SMMC7721细胞的杀伤作用。结果：透射电镜下可见T-exo为圆形或椭圆形双层膜囊泡状小体，大小不等，平均直径50～100 nm，表达外泌体膜相关分子及肿瘤抗原分子。负载T-exo 的DC 可促进初始T细胞增殖，T-exo致敏DC对AFP阳性的Huh-7肝癌细胞系杀伤活性明显高于AFP阴性的SMMC7721肝癌细胞组（ P＜0.05）及未负载T-exo 的对照组DC（ P＜0.05）。结论：负载T-exo 的DC 能促进T细胞增殖，提高CTL细胞的细胞毒活性诱导特异性的抗肝癌效应。经T-exo抗原致敏DC诱导的CTL对肝癌细胞有明显的细胞毒作用，明显高于DC对照组（ P＜0.05）。