目的：探索靶向MUC16的嵌合抗原受体修饰的NK-92（CARNK-92）细胞对卵巢癌的抗肿瘤活性。方法：通过免疫组化法及流式细胞术检测庆阳市中医医院妇产科卵巢癌外科手术15例患者的肿瘤组织及4种卵巢癌细胞系中MUC16的表达情况。通过全基因合成的方法合成 MUC CAR 序列并构建重组慢病毒表达载体，利用慢病毒感染的方式制备靶向 MUC16 的CARNK-92细胞（MUC-BBz）。通过流式细胞术检测MUC-BBz中CD107a的表达，以LDH释放法检测该工程细胞的激活及对靶细胞SKOV3的细胞毒性。建立SKOV3细胞裸鼠移植瘤模型，用来检测MUC-BBz的体内抗肿瘤活性。结果：卵巢癌患者肿瘤组织及人卵巢癌细胞中都高表达MUC16。通过慢病毒感染NK-92细胞成功构建MUC-BBz，其阳性率为（42.79±2.58）%。MUCBBz可以被过表达MUC16的肿瘤细胞特异性激活，与效靶细胞共孵育后，MUC-BBz中CD107a的表达量显著升高（P<0.01）、细胞因子IFN-γ以及穿孔素的分泌能力明显增加（均P<0.01）；LDH法检测显示，随着效靶比的增加，对4种卵巢癌细胞hey、COC1、SKOV3、A2780的杀伤作用均显著提高（均P<0.01）。体内移植瘤模型实验结果显示，输注 MUC-BBz 可以显著抑制裸鼠体内SKOV3细胞移植瘤的生长（P<0.01）。结论：构建的CARNK-92细胞可以在体内外显著抑制卵巢癌细胞的增殖，为进一步评估其临床治疗能力提供重要依据。

Objective: To explore the anti-tumor activity of MUC16-targeted chimeric antigen receptor modified NK-92 (CARNK-92) cells against ovarian cancer. Methods: The expression of MUC16 in surgically resected tumor tissues of 15 patients with ovarian cancer treated in the Department of Obstetrics and Gynecology of Qingyang Hospital of Traditional Chinese Medicine and 4 ovarian tumor cell lines was detected by Immunohistochemistry and Flow cytometry. MUC CAR sequence was synthesized by gene synthesis, and its lentivirus expression vector were constructed. CARNK-92 cells targeting MUC16 (MUC-BBz) were obtained by lentivirus infection. The expression of CD107a in MUC-BBz was detected by Flow cytometry. The activation of MUC-BBz cells and its cytotoxicity against SKOV3 target cells were characterized by the release of LDH assay. The xenograft nude mouse model of SKOV3 cells was established to verify the in vivo anti-tumor activity of MUC-BBz cells. Results: MUC16 was highly expressed in ovarian cancer tissues and human ovarian cancer cells. MUC-BBz was successfully constructed by infecting NK-92 cells with lentivirus, with a positive rate of (42.79±2.58)%. MUC-BBz could be specifically activated by MUC16 over-expressing tumor cells. After co-incubation of effector cells and target cells, the expression of CD107a on MUC-BBz was upregulated significantly (P<0.01), and the ability of MUC-BBz secreting cytokines IFN-γ and perforin also increased (all P<0.01). The LDH test indicated that with the increase of effector-target ratio, the cytotoxicity of MUC-BBz against 4 ovarian cancer cells (hey,COC1, SKOV3 andA2780) also significantly enhanced. The results of transplanted tumor model showed that transfusion of MUC-BBz could significantly inhibit the growth of SKOV3 xenograft in mice (P<0.01). Conclusion: The CARNK-92 cells can significantly inhibit the growth of ovarian cancer cells in vitro and in vivo, which provides an important basis for further evaluation of its clinical application.

国家自然科学基金资助项目（No.81260387）