目的：探讨使用CRSIPR/Cas9 技术删除CTL的免疫检查点CTLA-4 后CTL的抗裸鼠结肠癌移植瘤的效果及其作用机制。方法：针对CTLA-4 设计特异性小向导RNA(small guide RNA，sgRNA)并构建sgRNA/Cas9 质粒，使用慢病毒载体转染质粒至CTL获得删除CTLA-4 的CTL（CTLA-4 KOCTL），并验证质粒的转染效率和CTLA-4 的删除效率。BALB/c裸鼠随机分为2 组进行实验，预防性注射CTLA-4 KO CTL（实验组）及CTL（对照组），3 d 后接种结肠癌细胞株LS174-T，观察成瘤率及成瘤时间。建立结肠癌移植瘤裸鼠模型，成瘤后裸鼠随机分为2 组，分别给予CTLA-4 KO CTL（实验组）及CTL（对照组）进行细胞治疗，观察移植瘤的体积、裸鼠的生存时间，并检测移植瘤裸鼠血清中TNF-α 及IFN-γ 分泌水平。结果：设计sgRNA并成功构建以慢病毒为载体的CRSIPR/Cas9 系统，使用构建好的CRSIPR/Cas9 系统转染CTL细胞，转染效率最高可达（28.80±0.62)%，转染后以流式细胞术检测CTLA-4 的删除效率，CTLA-4 KO CTL 组的CTLA-4 表达较CTL 组显著降低[ （0.91±0.25）% vs（42.70±2.72）%，P<0.01]。预防实验中，实验组结肠癌移植瘤发生率明显低于对照组（33.33% vs 100.00%，P<0.01）。治疗实验中，实验组肿瘤体积较对照组显著减少[ （503.0±23.9）vs（911.2±51.4）mm3，P<0.05]，实验组裸鼠相较于对照组生存时间明显延长（中位生存期：78 vs 42 d，P<0.05），实验组裸鼠血清TNF-α（[ 268.93±17.04）vs（148.26±20.07）pg/ml，P<0.05]及IFN-γ（[ 315.38±18.67）vs (202.92±29.32) pg/ml，P<0.05]的分泌水平较对照组明显增高。结论：以CRSIPR/Cas9 技术成功删除CTL中免疫检查点CTLA-4 后可以显著抑制裸鼠结肠癌移植瘤的成瘤率、增强CTL对荷结肠癌裸鼠的抗肿瘤作用，并明显增高荷瘤鼠血清中TNF-α 和IFN-γ 水平。

Objective: To investigate the anti-tumor effect of CTL cells on colon cancer xenograft in nude mice after knocking out the immune check point CTLA-4 by CRISPR/Cas9 technology. Methods: A specific small guide RNA (sgRNA) for CTLA-4 was designed to construct sgRNA/Cas9 plasmid, which was then transfected into CTL using a lentiviral vector to obtain CTL cells with CTLA-4 deletion (CTLA-4 KO CTL). The transfection efficiency of the plasmid and the deletion efficiency of CTLA-4 were verified. BALB/c nude mice were randomly divided into two groups to prophylactically inoculate CTLA-4 KO CTL (experimental group) or CTL (control group); 3 days later, the animals of two groups were inoculated with colon cancer cell line LS174-T to observe the tumor formation rate and tumor formation time. After constructing colon cancer xenograft model in nude mice, the animals were randomly divided into two groups, respectively treated with CTLA-4 KO CTL (experimental group) and CTL (control group) cells to observe the tumor growth volume and survival time of mice. The serum levels of TNF-α and IFN-γ in nude mice were detected. Results: sgRNA was designed and CRSIPR/Cas9 system with lentivirus as vector was successfully constructed. CTL cells were transfected with the established CRSIPR/Cas9 system, and the highest transfection efficiency was up to (28.80±0.62)%. After transfection, the deletion efficiency of CTLA-4 was detected by Flow cytometry. The CTLA-4 expression of CTLA-4 KO CTL group was significantly lower than that of CTL group [(0.91±0.25)% vs (42.70±2.72)%, P<0.05]. In prophylactic assay, the formation rate of colon cancer xenografts in the experimental group was significantly lower than that in the control group (33.33%vs 100%, P<0.05). In treatment assay, the tumor volume in the experimental group was significantly inhibited compared with the control group ([503±23.9] vs [911.2±51.4] mm3, P<0.05), and the survival time of the experimental group was significantly prolonged (median survival time: 78 d vs 42 d, P<0.05); Moreover, the secretion levels of serumTNF-α ([268.93±17.04] pg/ml vs [148.26±20.07] pg/ml, P<0.05) and IFN-γ (315.38±18.67 pg/ml vs 202.92±29.32 pg/ml,P<0.05) in the experimental group were significantly higher than those in the control group. Conclusions: The lentiviral vector CRSIPR/Cas9 system is an effective gene editing method; its successful deletion of CTLA-4 in CTL cells can significantly inhibit the tumor formation rate of colon cancer xenografts in nude mice and enhance the anti-tumor effect of CTL on colon cancer xenografts.

河北省医学科学研究重点课题计划资助项目(No.20180375)