目的：探讨CD3和CD28单克隆抗体联合作用对细胞因子诱导的杀伤(cytokine-induced killer，CIK)细胞及其亚群增殖及功能的影响。方法：采集2014年6月至2015年6月在郑州大学第一附属医院接受生物细胞治疗的20例肿瘤患者（肾癌6例，肺癌5例，肝癌、乳腺癌和恶性黑素瘤各2例，胆囊癌、淋巴瘤和卵巢癌各1例）的外周血，每例外周血均分为4组（对照组，单用CD3单抗组，单用CD28单抗组，CD3和CD28单抗联合作用组）进行CIK细胞的诱导培养。用CFSE染色检测CIK细胞的增殖能力；ELISA检测CIK细胞分泌细胞因子IFN-γ、TNF-α和IL-2的水平；磁珠分选CIK细胞亚群CD4+、CD8+和CD56+细胞，然后用ELISA检测对CIK细胞亚群分泌IFN-γ、TNF-α和IL-2能力的影响。结果： CD3/CD28单抗联合作用（PI=5935）对CIK细胞的增殖能力的影响与单独使用CD3单抗（PI=64.4）相比效果并不明显，但是可以刺激CIK细胞分泌较高水平的IFN-γ和TNF-α\[IFN-γ: (384.6±2631) vs (201.5±271.5) pg/ml, P=0.0361; TNF-α: (4.795±1.251) vs (2.835±0.443) pg/ml, P=0.0265\]；进一步分析发现，两单抗的联合作用可增强亚群细胞CD8+分泌IFN-γ、CD56+分泌IL-2和TNF-α 的活性，从而起到对淋巴细胞功能的增强作用。结论： CD3与CD28单抗联合作用不能增强CIK的增殖能力,但可以在一定程度上增强CIK细胞的功能，在肿瘤的细胞免疫治疗应用中具有一定的潜力。

Objective:To explore the effect of combined action of anti-CD3 and anti-CD28 monoclonal antibodies on the proliferations and functions of cytokine-induced killer (CIK) cell and its subsets. Methods: Twenty peripheral blood samples of the patients with tumors (6 cases of renal carcinoma, 5 cases of lung cancer, every 2 cases of hepatic carcinoma, breast cancer and melanoma, and every 1 case of gallbladder carcinoma, lymphoma and ovarian cancer) who received biotherapy at the First Hospital Affiliated to Zhengzhou University from June 2014 to June 2015 were collected. Each sample of the peripheral blood was divided into four groups on average, namely control group, anti-CD3 monoclonal antibody alone group, anti-CD28 monoclonal antibody alone group and anti-CD3 combined with anti-CD28 monoclonal antibodies group, which were cultured with CIKs. Proliferation of the CIK cells was detected with CFSE staining. Secretion levels of IFN-γ, TNF-α and IL-2 by the CIK cells were detected by ELISA. CD4+, CD8+ and CD56+ cells of CIK cell subsets were sorted with magnetic beads. Then their abilities of secreting IFN-γ, TNF-α and IL-2 were examined by ELISA assay. Results:No significant difference in the proliferation ability of the CIK cells was detected between the anti-CD3/anti-CD28 combined group (PI=59.35) and anti-CD3 alone group (PI=64.4). However, combination of anti-CD3 and anti-CD28 monoclonal antibodies could stimulate CIK cells to secrete higher level of IFN-γ and TNF-α than those done by anti-CD3 alone, IFN-γ: (384.6±263.1) pg/ml vs (201.5±271.5)pg/ml, P=0.0361; TNF-α (4.795±1.251)pg/ml vs (2.835±0.443)pg/ml, P=0.0265. Furth analysis found that the combined action of anti-CD3 and anti-CD28 monoclonal antibodies could enhance activity of CD8+ cell subset to secret IFN-γ and activity of CD56+ cell subset to secret IL-2, thus strengthen the functions of lymphocytes. Conclusion: The combined action of anti-CD3 and anti-CD28 monoclonal antibodies could not enhance proliferation of the CIK cells, but strengthen function of the CIK cells to a certain extent, which has certain potential to be used in the immunotherapy of tumor cells.

国家自然科学基金面上项目(No.81271815,81171986); 国家自然科学基金中美生物医学合作研究项目(No.812111102);卫生部科研攻关基金资助项目(No.20110110001)；河南省科技厅基础与前沿技术研究基金(No.112300410153,122300410155)