目的：评价新建立的K562工程细胞联合IL-2扩增方案对人NK细胞扩增和活化的效果。 方法：采集健康志愿者和肿瘤患者的外周血PBMC并分离NK细胞，采用前期构建的K562工程细胞(将IL-15、4-1BBL和IL-18在白血病K562细胞上进行跨膜表达获取）联合IL-2培养方案对NK细胞进行扩增和活化，以流式细胞术检测NK细胞的扩增效果和NK细胞表面受体表达水平，CCK-8法检测扩增后NK细胞对肿瘤细胞的杀伤活性和ADCC活性，CCK-8法检测在培养方案扩增末期加入TKD多肽对NK细胞的活化效果。结果：对于健康志愿者的NK细胞，新建立扩增培养方案可使NK细胞在PBMC中的比例提高至（93±3）%；使NK细胞中活化性受体NKG2D、CD94、NKp30、NKp44和NKp46的比例分别提高60%、40%、20%、40%和63%，而抑制性受体的表达变化不大；扩增后NK细胞对白血病细胞K562、肺癌细胞A549、肝癌细胞SMMU-7721和乳腺癌细胞MCF-7的杀伤活性分别提高了19%、29%、26%和28%，其ADCC活性从（33±5.6）%上升至（65±12）%；方案中增加TKD可使NK细胞的杀伤活性从（86±4）%提高至（96±2）%。对于肿瘤患者的NK细胞，新扩增方案使其在PBMC的比例提高至（90.0±8.0）%，其对K562细胞的杀伤活性提高了17%左右。 结论： K562工程细胞联合IL-2扩增方案可高效扩增NK细胞，明显激活其杀伤活性，扩增和活化的NK细胞可满足临床治疗的需要。

Objective: To evaluate the effects of K562 cells overexpression of IL-15, 4-1BBL and IL-18 combined with IL-2 ex vivo expansion on NK cell activation and cytotoxicity. Methods: K562 cells were engineered to overexpress IL-15, 4-1BBL and IL-18 on the cell membrane. Peripheral blood mononuclear cells (PBMCs) were prepared from venous blood collected from both healthy volunteers and cancer patients. NK cells were purified from PBMCs and expanded ex vivo by co-culture with engineered K562 cells in the presence of IL-2 and/or Hsp70 peptide (TKD) for 21 days. The purity and surface marker expression of the expanded NK cells were analyzed by flow cytometry. The cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) of the expanded NK cells were assessed by a Cell Counting Ki-8-based colorimetric assay. Results: In the cell preparations from healthy subjects, ex vivo expansion for 3 weeks by co-culture with engineered K562 cells in the presence of IL-2 and/or TKD increased the purity of NK cells to (93±3)%, increased the proportion of cells expressing killer activation receptors, NKG2D, CD94, NKp46, NKp30, and NKp44, by 60%, 40%, 20%, 40%, and 63% respectively with no effect on the proportion of cells expressing inhibitory receptors (CD158b, NKB1 and NKAT2), increased the ADCC by 32%, and increased the cytotoxicity against K562, A549, 7721, MCF tumor cells by 19%, 29%, 26% and 28% respectively. In the cell preparations from cancer patients, ex vivo expansion induce the percentage of NK cells increase to (90.0±8.0)% and the cytotoxicity against K562 cells by 17%.Conclusion: Co-culture with engineered K562 cells overexpressing IL-15, 4-1BBL and IL-18 in the presence of IL-2 and/or TKD may offer an effective approach to expand the NK cell population ex vivo and enhance the NK cell cytotoxicity.

陕西省自然科学基金资助项目（No. 2014JM4171）