目的：构建表达CD19 的二代CAR-NK-92 细胞系，探讨其对CD19 阳性非霍奇金淋巴瘤细胞的特异性杀伤作用。方法：构建表达CD19-CAR基因的载体并包装慢病毒颗粒，感染NK-92 细胞，流式细胞术检测感染率，并进一步分选阳性细胞，Western blotting 检测CD19-CAR 在NK-92 细胞中的表达；以CD19 阴性骨髓瘤细胞U-266、CD19 阳性的非霍奇金淋巴瘤细胞ARH-77 和HS-Sultan 细胞为靶细胞，以CD19CAR-NK-92 为效应细胞，流式细胞术检测细胞杀伤实验中存活肿瘤细胞的绝对数量并计算杀伤率。结果: 成功构建慢病毒载体pLVX-CD19-CAR并包装病毒颗粒，感染NK-92 细胞后CD19CAR-NK-92 细胞纯度高于90%；Western blotting 分析显示CD19-CAR已经成功表达在NK-92 中。CD19CAR-NK-92 对ARH-77[ （70.10±1.86）% vs （1.95±0.63）%，P<0.01]和HS-Sultan[ （74.98±1.60）% vs（0.58±1.49）%，P<0.01]细胞的杀伤率显著高于空载体对照组ZsGreen-NK-92 细胞，对U266 的杀伤率无显著差别（P>0.05）。结论：成功构建了表达CD19CAR的NK-92 细胞系，其对CD19 阳性的非霍奇金淋巴瘤细胞有特异性杀伤作用。

Objective: A second generation CAR-NK-92 cell line expressing CD19 was constructed to investigate its specific killing effect on CD19 positive non-Hodgkin lymphoma cells. Methods: First, build CD19-CAR gene expression vector and packaged slow virus particles, then the infection rate was detected by flow cytometry after infected NK-92 cells and positive cells were further separated.Finally, detected the expression of CD19-CAR in NK-92 cells by Western blotting. U-266 with CD19 negative myeloma cells, ARH-77 and HS-Sultan with CD19 positive non-Hodgkin ’ s lymphoma cells as target cells, and CD19CAR-NK-92 as effector cells, then the killing rate was calculated by the absolute number of tumor cells alive in the cell killing experiment. Results: Construct lentivirus vector pLVX-CD19-CAR and packaged virus particles successfully, the purity of CD19-CAR-NK-92 cells also was over 90% after infected with NK-92 cells; and Western blotting analysis showed that CD19-CAR had been successfully expressed in NK-92 cell. The killing effect of CD19CAR-NK-92 on ARH-77 ([70.10±1.86]% vs [1.95±0.63]%, P<0.01) and HS-Sultan ([74.98±1.60]% vs [0.58±1.49]%, P<0.01) cells was significantly higher than the empty vector control group of ZsGreen-NK-92, but there was no difference in killing U266 (P>0.05). Conclusion: The NK-92 cell lines expressing CD19CAR were successfully constructed, and also has specific killing effects on CD19 positive non-Hodgkin lymphoma cells.

浙江省重点科技创新团队资助项目（No. 2011R50018-06）