摘要
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 KO CTL),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)mm^3,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] mm^3, 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 serum TNF-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.
作者
师龙
耿耸松
蔡子琪
韩锦胜
赵咫龙
张伟
宋鸿涛
孟桐羽
蔡建辉
SHI Long;GENG Songsong;CAI Ziqi;HAN Jinsheng;ZHAO Zhilong;ZHANGWei;SONG Hongtao;MENG Tongyu;CAI Jianhui(Department of Vascular Surgery,the Second Hospital of Hebei Medical University,Shijiazhuang 050000,Hebei,China;Nongfuyu(Hebei)Biomedical Co.,Ltd.,Shijiazhuang 050000,Hebei,China;Department of Gastroenteroabdominal Hernia,Cangzhou Hospital of Traditional Chinese and Western Medicine,Cangzhou 061000,Hebei,China;Department of Hepatobiliary Surgery,the Third Affiliated Hospital of Jinzhou Medical University,Jinzhou 121000,Liaoning,China;The Second Department of General Surgery,Handan Central Hospital,Handan 056001,Hebei,China;Department of Peripheral Vascular Disease,the Second Hospital of Shijiazhuang,Shijiazhuang 050000,Hebei,China;Department of Gynecology,Shijiazhuang People's Hospital,Shijiazhuang 050000,Hebei,China;General Surgery&Oncology Department,the Hebei General Hospital,Shijiazhuang 050000,Hebei,China)
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2020年第3期221-227,共7页
Chinese Journal of Cancer Biotherapy
基金
河北省医学科学研究重点课题计划资助项目(No.20180375)。
