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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 investigate the activation of human antigen presenting cell (aAPC) K32 / 4-IBBL / CD86 on CD8 + T cells of hepatocellular carcinoma. Methods: CD8 + T cells were isolated from peripheral blood of patients with HLA-A2-positive hepatocellular carcinoma by magnetic beads method. The aAPC and CD8 + T cells were mixed and cultured in different ratios (1: 1, 1: 2, 1: 3) to induce CTL. Trypan blue exclusion assay was used to determine the growth curve of CTL. MTS / PMS assay was used to detect the proliferation of CTL. Flow cytometry was used to detect the ability of CTL to secrete IFN-γ. MTT assay was used to detect the cytotoxicity of CTL on human hepatocellular carcinoma cell line BEL7402 and autologous hepatoma cells effect. Results: The proliferative ability of CD8 + T cells in peripheral blood of patients with hepatocellular carcinoma was significantly increased after aAPC treatment. The proportion of CD8 + T cells was (21.2 ± 2.5) × 105 on the 8th day after mixed with 1: 1, 1: 2 and 1: (P <0.01). The proportion of CTL secreting IFN-γ after mixed culture were (26.2 ± 1.91), (17.6 ± 3.2) × 105 and (15.3 ± 2.8) × 105 ), (21.3 ± 1.38)%, (18.6 ± 1.20)% respectively, which was significantly higher than that of the control group (0.1 ± 0.02)% (P <0.01). The cytotoxicity of CTLs activated by aAPC on BEL7402 cells and autologous hepatocarcinoma cells was significantly increased compared with that of the control cells. The cytotoxicity of CTLs against BEL7402 cells and autologous hepatocarcinoma cells was (21.8 ± 4.3)% and ( (3.8 ± 2.0)% and (3.8 ± 2.0)%, respectively (P <0.01). When CTL was 1:50 in the ratio of 50: 1, CTL of BEL7402 cells (56.8 ± 3.0)% and the rate of autophagic cell killing (64.8 ± 4.2)% were significantly higher than that of the 1: 2 mixed culture group (44.3 ± 2.6), (56.1 ± 3.4)% and 1: 3 The mixed culture group (38.9 ± 4.7)%, (46.2 ± 4.7)% (P <0.05). CONCLUSION: aAPC can efficiently activate CD8 + T cells in vitro and induce IFN-γ production in CTLs. In addition, CTL has obvious cytotoxicity on human hepatocellular carcinoma cell line BEL7402 and autologous hepatocarcinoma cells.