靶向前列腺癌CAR-NK细胞的抗肿瘤活性评价<sup>*</sup>

doi:10.13523/j.cb.2302030

中国生物工程杂志

2023, Vol. 43

Issue (6): 1-11 DOI: 10.13523/j.cb.2302030

研究报告

靶向前列腺癌CAR-NK细胞的抗肿瘤活性评价^*

苏凌宇¹,邹金桃¹,牛安娜¹,张伟²,张晓鹏^1,^**(

),陈薇^1,^**(

)

1 军事科学院军事医学研究院北京 100071
2 南湖实验室嘉兴 314001

Anti-tumor Effect of CAR-NK Cells Targeting Prostate Cancer

SU Ling-yu¹,ZOU Jin-tao¹,NIU An-na¹,ZHANG Wei²,ZHANG Xiao-peng^1,^**(

),CHEN Wei^1,^**(

)

1 Academy of Military Medical Sciences, Beijing 100071,China
2 Nanhu Laboratory,Jiaxing 314001,China

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摘要：

目的:探索基于DAP12共刺激信号和靶向前列腺干细胞抗原(PSCA)的嵌合抗原受体NK细胞(CAR-NK)对前列腺肿瘤细胞的杀伤作用。方法:使用慢病毒转染系统构建CAR-NK细胞,使用流式细胞术检测前列腺癌细胞DU145 PSCA的表达水平和CAR-NK细胞的阳性率,并经细胞和动物模型评价CAR-NK细胞的抗肿瘤活性。结果:前列腺癌细胞DU145高表达PSCA,阳性率约为98.50%。流式细胞术检测显示CAR-NK细胞CAR分子表达阳性率为(64.07 ± 3.01)%。细胞毒性实验发现,与对照NK细胞相比,携带DAP12共刺激信号的CAR-NK细胞具有较强抗前列腺肿瘤作用,杀伤率提升了约1.5倍。ELISA结果显示,与对照NK细胞相比,CAR-NK细胞杀伤DU145细胞时释放的TNF-α、IFN-γ、CD107α、Granzyme B和Perforin-1等因子水平显著提高。动物实验表明,CAR-NK细胞相对于对照NK细胞,更能有效抑制肿瘤增殖,两者之间有显著性统计学差异(P<0.000 1)。结论:靶向PSCA并提供DAP12共刺激信号的CAR-NK细胞具有较强杀伤前列腺肿瘤的作用,为前列腺癌治疗提供了潜在的细胞药物。

关键词： CAR-NK; PSCA; DAP12; 前列腺癌

Abstract:

Objective: To investigate the anti-tumor efficacy of chimeric antigen receptor-nature killing cells (CAR-NK cells) against prostate stem cell antigen (PSCA) via DAP12 co-stimulation signal. Methods: CAR-NK cells were generated via lentiviral transfection and the expression of CAR in NK cells was evaluated using flow cytometry. The anti-tumor activity of CAR-NK cells was further assessed in both cellular and animal models. Results: The prostate cancer cell DU145 expressed PSCA with a positive rate of about 98.50%. Flow cytometry revealed that (64.07 ± 3.01)% of CAR-NK cells were positive. Furthermore, the killing rate of CAR-NK cells was approximately 1.5 times higher than that of control NK cells. ELISA tests showed that CAR-NK cells released significantly higher levels of TNF-α, IFN-γ, CD107α, Granzyme B, and Perforin-1, when compared to control NK cells in the process of killing DU145 cells. Additionally, animal experiments show that CAR-NK cells had a better inhibiting effect in the growth of DU145 tumor cells in mice compared with control NK cells as significant difference was found between the two groups (P<0.000 1). Conclusion: The findings suggested that PSCA-targeting CAR-NK cells with DAP12 had a greater anti-prostate tumor effect, providing a potential cellular therapeutic option for the treatment of prostate cancer.

Key words: CAR-NK PSCA DAP12 Prostate cancer

收稿日期: 2023-02-17 出版日期: 2023-07-04

ZTFLH:

Q819

基金资助: * 国家自然科学基金(82173787)

通讯作者: **电子信箱:zxp8565@aliyun.com;cw0226@foxmail.com

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引用本文:

苏凌宇, 邹金桃, 牛安娜, 张伟, 张晓鹏, 陈薇. 靶向前列腺癌CAR-NK细胞的抗肿瘤活性评价^*[J]. 中国生物工程杂志, 2023, 43(6): 1-11.

SU Ling-yu, ZOU Jin-tao, NIU An-na, ZHANG Wei, ZHANG Xiao-peng, CHEN Wei. Anti-tumor Effect of CAR-NK Cells Targeting Prostate Cancer. China Biotechnology, 2023, 43(6): 1-11.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2302030 或 https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I6/1

图1 基于DAP12信号域的CAR质粒的设计与构建

图2 慢病毒滴度检测

图3 流式细胞术检测CAR的表达水平

图4 流式细胞术检测DU145细胞PSCA表达水平

图5 CAR-NK细胞的杀伤能力

图6 ELISA法测定细胞因子含量

图7 小鼠体内肿瘤负荷检测

图8 CAR-NK与NK细胞浸润情况

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