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目的:建立从20mL外周血中高效扩增抗CD3单克隆抗体激活的杀伤细胞(CD3AK)的培养体系,并进行体外细胞毒活性检测。方法:采用抗CD3单克隆抗体(Anti-CD3mAb)激活人外周血单个核细胞(PBMC),在人重组白细胞介素2(rIL-2)的协同活化下,获得CD3AK。对CD3AK进行长期体外培养,进行增殖动力学、免疫表型和体外抗肿瘤活性的分析。结果:在培养第7~22天,CD3AK增殖倍数明显提高,最高可达317倍;免疫表型分析显示,CD3+细胞从活化前的51.9%增加至94.6%,CD4+细胞从19.5%增加至51.1%,CD8+细胞从22.1%增加至52.1%;效靶比20∶1组和40∶1组的杀伤肿瘤细胞活性明显高于10∶1组(P<0.05和P<0.01);在7~19 d的培养天数之内,杀伤肿瘤细胞活性最强。结论:从外周血中高效扩增的CD3AK是以CD3+、CD4+和CD8+细胞为主的异质性细胞群,在体外有明显的杀伤肿瘤细胞活性。
OBJECTIVE: To establish a culture system that efficiently expand anti-CD3 monoclonal antibody-activated killer cells (CD3AK) from 20 mL of peripheral blood and detect the cytotoxicity in vitro. Methods: Human peripheral blood mononuclear cells (PBMCs) were activated by anti-CD3 mAb and CD3AK was obtained by synergistic activation of human recombinant interleukin-2 (rIL-2). CD3AK long-term in vitro culture, proliferation kinetics, immunophenotype and in vitro anti-tumor activity analysis. Results: The multiplication of CD3AK was significantly increased 317 days after culture on day 7-22. The immunophenotype analysis showed that CD3 + cells increased from 51.9% to 94.6% and CD4 + cells increased from 19.5% to 51.1% , And the percentage of CD8 + cells increased from 22.1% to 52.1%. The cytotoxic activity of target cells in 20: 1 and 40: 1 groups was significantly higher than that in 10: 1 group (P <0.05 and P <0.01) Within the training days, the strongest killing tumor cells. CONCLUSION: CD3AK, highly efficient from peripheral blood, is a heterogeneous cell population mainly composed of CD3 +, CD4 + and CD8 + cells, which has obvious killing activity on tumor cells in vitro.