目的：比较AFP抗原以不同方式负载DCs对后者生物学功能的影响及体外刺激CTL对肝癌HepG2细胞的杀伤作用。方法：分离健康供者外周血单个核细胞培养DC，分别用（A）人工合成AFP抗原肽、（B）HepG2细胞裂解物、（C）HepG2细胞分泌的外泌体（tumor exosome, T-exo）、（D）AFP抗原的重组腺相关病毒（recombinant adeno-associated virus expressing α-fetoprotein antigen, rAAV/AFP）致敏DC前体细胞及（E）未负载抗原的DC作为对照，经GM-CSF、IL-4及LPS联合诱导DCs分化成熟。流式细胞术检测rAAV/AFP病毒感染效率、各组DCs表型及其剌激初始T细胞的增殖效应，7-ADD/CFSE双染法流式细胞术检测各组DCs诱导CTL对AFP阳性HepG2细胞的杀伤作用。结果：几种抗原负载方式均可诱导DCs成熟、促进CTL增殖及特异性识别并杀伤HepG2细胞，但rAAV/AFP感染DCs后，其CD83、CD86、ICAM-1、CD58、CD40分子表达水平明显高于对照组（P<0.05），rAAV/AFP+DC组和HepG2-Texo+DC组对HepG2细胞的杀伤作用均分别显著优于其他抗原负载（AFP/peptide+DC、HepG2 lysate+DC）组\[（44.92±4.12）% vs（28.42±3.29）%、（24.28±1.79)%;（41.40±2.87）% vs （28.42±3.29）%、（24.28±1.79）%；均P<0.05）\]。结论：rAAV/AFP高效感染DCs后能有效刺激初始T细胞增殖，并增强CTL对AFP阳性靶细胞的杀伤活性，而负载Texo的DCs也能诱导显著的抗肝癌效应，上述结果为基于DCs的肝癌疫苗的研发提供新的思路。

Objective:To evaluate the cytotoxic effects of the cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) loaded with alpha-fetoprotein (AFP) in different approaches. Methods:Peripheral blood monocytes were isolated from healthy donors. The adhesive precursor DCs were cultured in the presence of rhGM-CSF and rhIL-4 for 6 d. The cytokine-treated DCs were then left untreated as a control, infected with recombinant AFP-carrying adeno-associated viral vector (rAAV/AFP), or loaded respectively with AFP peptide, HepG2 cell lysate, and HepG2-derived exosomes (T-exo). Changes in CD83, CD86, ICAM-1,CD58 And CD 40 in DCs before and after AFP loading were assessed by Western botting Autologous T cells were co-cultured with the various AFP-loaded DCs loaded at a ratio of 10∶1 for 48 h. The proliferative activity and cytotoxicity against HepG2 cells of DC-induced T cells were assessed by flow cytometry.Results: AFP antigen induced maturation of DCs. The expression of surface molecules CD83,CD86, ICAM-1,CD58 and CD40 in DCs infected with the rAAV/AFP vector was significantly increased compared with the nave DCs (P<0.05). AFP-loaded DCs increased the proliferation of T cells. The cytotoxic activity against HepG2 cells was (41.40±2.87)%, (44.9±4.12)%, (28.42±3.29)%, and (24.28±1.79)% for T cells after induction by T-exo-loaded DCs, rAAV/AFP-infected DCs, AFP peptide-loaded DCs and HepG2 lysate-treated DCs respectively (P<0.05). Conclusions：AFP and T-exsome are capable of increasing the proliferative activity of DCs and the cytotoxic activity of CTL against HepG2 cells. Our finding may have significant implications in the development of DC-based vaccines for liver cancer.

国家卫生和计划生育委员会科研基金——福建省卫生教育联合攻关计划资助项目（No. WKJ-FJ-04）；厦门大学药学院-福建省肿瘤医院促进科技合作联合研究基金资助项目（No. 2014LY-2L-04）