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目的研究激发型CD28单抗参与诱导的CIK的扩增能力及细胞表型特征。方法采用Ficoll分离法获取健康人外周血单个核细胞(PBMC),调整浓度为2×106/mL后,接种于24孔培养板,1mL/孔。按以下2组添加诱导剂:1)抗-CD3+IL-2+IFN-γ;2)抗-CD3+抗-CD28+IL-2+IFN-γ。诱导剂的终浓度抗-CD3为1μg/mL、抗-CD28为0.5μg/mL、IL-2为0.1万U/mL、IFN-γ为100ng/mL。隔2d换液,培养至d7,经全自动血细胞计数仪计算各孔细胞总量。采用免疫荧光单标记法分析CIK表面CD4+、CD8+及CD56+的表达,免疫荧光双标记法分析CIK表面CD4+/CD25+、CD8+/CD25+、CD4+/FasL+、CD8+/FasL+的表达。结果培养至d7,激发型CD28单抗组CIK的细胞总量为(78.2±7.3)×106/孔,明显高于对照组的(27.8±2.7)×106/孔,差异具有统计学意义(P<0.05)。单标记法的分析结果显示,激发型CD28单抗参与的CIK中CD4+细胞、CD8+细胞及CD56+细胞分别为(68.4±6.1)%、(34.6±7.2)%及(15.1±3.9)%,与对照组的(52.5±3.6)%、(26.9±2.7)%及(7.7±1.2)%比较均具有显著统计学差异(P<0.05)。双标记法的分析结果显示,激发型CD28单抗参与诱导的CIK细胞中CD4+FasL+T细胞、CD8+FasL+T细胞、CD4+CD25+T细胞及CD8+CD25+T细胞比例分别为(36.6±4.7)%、(40.7±3.2)%、(60.1±5.3)%及(58.5±4.1)%。与对照组的(21.9±3.9)%、(24.3±5.1)%、(45.6±4.6)%及(44.6±3.4)%比较均具有统计学意义(P<0.05)。结论激发型CD28单抗可增强CIK的扩增能力及上调细胞活化膜型分子的表达,提示激发型CD28单抗可具有提高CIK杀瘤能力。
Objective To study the ability of proliferation and cell phenotype of CIK induced by CD28 monoclonal antibody. Methods Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll separation. After adjusting the concentration to 2 × 106 / mL, the cells were inoculated into 24-well culture plates at 1 mL / well. Inducers were added according to the following 2 groups: 1) anti-CD3 + IL-2 + IFN-γ; 2) anti-CD3 + anti-CD28 + IL-2 + IFN-γ. The final concentration of inducer was 1 μg / mL for anti-CD3, 0.5 μg / mL for anti-CD28, 0.1 million U / mL for IL-2 and 100 ng / mL for IFN-γ. Every 2d fluid exchange, cultured to d7, by the automatic hemocytometer to calculate the total amount of cells in each well. The expression of CD4 +, CD8 + and CD56 + on CIK surface was analyzed by immunofluorescence single labeling. The expression of CD4 + / CD25 +, CD8 + / CD25 +, CD4 + / FasL +, CD8 + / FasL + on CIK surface was analyzed by immunofluorescence double labeling. Results The total number of CIK cells in the challenged CD28 mAb group was (78.2 ± 7.3) × 106 / well, which was significantly higher than that in the control group (27.8 ± 2.7) × 106 / well, the difference was statistically significant (P <0.05). The results of single-marker analysis showed that CD8 + cells and CD56 + cells in CIK were (68.4 ± 6.1)%, (34.6 ± 7.2)% and (15.1 ± 3.9)%, respectively, (52.5 ± 3.6)%, (26.9 ± 2.7)% and (7.7 ± 1.2)% respectively. There was a significant difference between the two groups (P <0.05). The results of double-labeling assay showed that the proportion of CD4 + FasL + T cells, CD8 + FasL + T cells, CD4 + CD25 + T cells and CD8 + CD25 + T cells in induced CIK cells were 36.6 ± 4.7%, (40.7 ± 3.2)%, (60.1 ± 5.3)% and (58.5 ± 4.1)%, respectively. (21.9 ± 3.9)%, (24.3 ± 5.1)%, (45.6 ± 4.6)% and (44.6 ± 3.4)% respectively in the control group (P <0.05). Conclusion CD28 monoclonal antibody can enhance the ability of CIK amplification and up-regulate the expression of activated cell membrane molecules, suggesting that CD28 monoclonal antibody may enhance CIK killing ability.