目的: 探讨人工抗原提呈细胞（artificial antigen presenting cell，aAPC）K32/4-IBBL/CD86对肝癌CD8+T细胞的活化作用。方法: 磁珠法分选HLA-A 2阳性肝癌患者外周血CD8+T细胞，aAPC与CD8+T细胞按不同比例（1∶1、1∶2、1∶3）混合培养，诱导CTL。锥虫蓝拒染法测定CTL的生长曲线，MTS/PMS法检测CTL的增殖，流式细胞术检测CTL分泌IFN-γ的能力，MTT法检测CTL对人肝癌细胞株BEL7402和自体肝癌细胞的杀伤作用。结果: 肝癌患者外周血CD8+T细胞经aAPC作用后，增殖能力明显增强，按1∶1、1∶2、1∶3比例混合培养后第8天分别为（21.2±2.5）×105、（17.6±3.2）×105、（15.3±2.8）×105，明显高于对照组的（8.5±0.15）×105（P<0.01），混合培养后分泌IFN-γ的CTL比例分别为（26.2±191）%、（21.3±1.38）%、（18.6±1.20）%，明显高于对照组的（0.1±0.02）%（P<0.01）。aAPC活化的CTL对BEL7402细胞和自体肝癌细胞的杀伤率较对照组显著增强，按1∶3混合培养后得到的CTL对BEL7402细胞和自体肝癌细胞的杀伤率分别为（21.8±43）%和（25.6±3.6）%，明显高于对照组的（3.8±1.8）%和（3.8±2.0）%（P<0.01）；效靶比为50∶1时，1∶1混合培养组CTL对BEL7402细胞的杀伤率（56.8±3.0）%和对自体肝癌细胞的杀伤率（64.8±4.2）%明显高于1∶2混合培养组的（44.3±2.6）%、（56.1±3.4）%和1∶3混合培养组的（38.9±4.7）%、（46.2±4.7）%（P<0.05）。结论: aAPC在体外可高效活化肝癌患者CD8+T细胞，诱导CTL分泌IFN-γ，且CTL对人肝癌细胞株BEL7402和自体肝癌细胞具有明显的杀伤作用。

Objective:To explore the activation of CD8+T cells of hepatocellular carcinoma (HCC) stimulated by the artificial antigen presenting cells (aAPC) (K32/4-IBBL/CD86). Methods: Peripheral CD8+T cells of HLA-A*0201 positive HCC patients were enriched by MACS separation system. CD8+T cells were cultured with aAPC at different ratios (3∶1, 2∶1 and 1∶1), and CTLs were induced. Growth curve of CTLs was examined by trypan blue exclusion assay; MTS/PMS assay was used to detect the proliferation of CTLs; the IFN-γ secreting CTLs were assayed by FACS; the cytotoxicity of CTLs on a human liver cancer cell line BEL7402 and self-HCC cells was tested using MTT method. Results: The proliferation of peripheral CD8+ T cells stimulated with aAPC was significantly increased. At days 8, the CTL cell numbers of aAPC to CD8+ T cells at 1∶1, 1∶2, 1∶3 groups was significantly higher than those in the control group (\[212±2.5\]×105, \[17.6±3.2\]×105, \[15.3±2.8\]×105 vs \[8.5±0.15\]×105, P<0.01); and the proportion of IFN-γ secreting CTLs in 1∶1, 1∶2, 1∶3 groups was higher than those in the control group (\[26.2±1.91\]%, \[21.3±1.38\]%, \[18.6±1.20\]% vs \[0.1±0.02\]%, P<0.01). The cytotoxicity of aAPC-activated CTLs against BEL7402 cells and self-HCC cells in 1∶3 group was stronger than that in the control group (\[21.8±4.3\]% vs \[3.8±18\]%, P<0.01; \[25.6±3.6\]% vs \[3.8±2.0\]%, P<0.01). A higher cytotoxic rate on BEL7402 cells and self-HCC cells in the 1∶1 group at a ratio of E (effect): T (target) as 50∶1 was achieved compared with that in the 1∶2 and 1∶3 groups (\[568±3.0\]% vs \[44.3±2.6\]%, \[38.9±4.7\]%, P<0.05; \[64.8±4.2\]% vs \[56.1±3.4\]%, \[46.2±47\]%, P<0.05). Conclusion: aAPC can effectively activate CD8+T cells of HCC patients, and the resultant CTLs not only secrete IFN-γ but also efficiently kill human liver cancer BEL7402 cells and self-HCC cells.

江苏省卫生厅预防医学科研基金资助项目（No.Y201032）