构建靶向血管内皮生长因子受体2可调控活性的嵌合抗原受体T细胞及其体外功能验证  

Construction of a chimeric antigen receptor T cell targeting vascular endothelial growth factor receptor 2 and its in vitro function verification

作　　者：杨超[1] 郑勇斌[1] 童仕伦[1] 何小波[1] 曹峰瑜[1] 黄朔阳 黎华丽 程煌荣 Yang Chao;Zheng Yongbin;Tong Shilun;He Xiaobo;Cao Fengyu;Huang Shuoyang;Li Huali;Cheng Huangrong(Department of Gastrointestinal Surgery,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院胃肠外1科,武汉430061

出　　处：《中华实验外科杂志》2021年第2期263-265,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金项目 (81372553);吴阶平医学基金会临床科研专项资助基金 (320.2710.1855)。

摘　　要：目的构建靶向血管内皮生长因子受体2(VEGFR2)可调控活性的小分子门控嵌合抗原受体T(smgCAR-T)细胞系统并在体外验证其抗肿瘤和可调节活性。方法采用基因全长合成和磷酸钙转染法构建smgCAR-T及其阳性(CAR+-T)和阴性(CAR--T)对照细胞。同时构建表达VEGFR2的小鼠结直肠癌细胞(MCA38VEGFR2+)。验证并筛选构建成功的细胞后再体外验证CAR-T细胞靶向结合能力。在不同的效靶比下检测白细胞介素-2和肿瘤坏死因子-γ的浓度来评估CAR-T细胞的体外分泌功能,用乳酸脱氢酶浓度评估CAR-T细胞的体外抗肿瘤活性。组间的比较采用t检验或单因素方差分析。结果基因测序和流式细胞术证实CAR-T细胞和靶细胞构建成功。与对照组比较,CAR+-T细胞+MCA38VEGFR2+组具有最强的靶细胞杀伤能力。在smgCAR-T细胞+MCA38VEGFR2+组中,随着C16-(S)-3-甲基吲哚雷帕霉素(Rapalog)滴度升高靶细胞杀伤率逐渐升高;当Rapalog为40 nmol/L时杀伤率达到最大值,为(66.25±13.20)%。分别向smgCAR-T细胞+MCA38VEGFR2+细胞组(A和B组)加入40 nmol/L的Rapalog后,在24 h时可观察到两组杀伤率出现同步升高[(44.25±6.24)%比(49.25±5.56)%,t=1.197,P>0.05],差异无统计学意义。在置换培养基后,再次添加了Rapalog的A组中观察到靶细胞杀伤率升高明显;而未再次添加Rapalog的B组中靶细胞杀伤率升高缓慢;此时,A和B组杀伤率差异有统计学意义[(89.00±3.16)%比(56.50±6.61)%,t=8.873,P<0.01]。结论构建的smgCAR-T细胞在体外展现出一定的抗肿瘤活性,且其活性受小分子化合物Rapalog的可逆调控。Objective To construct a small molecular gated chimeric antigen receptor T(smgCAR-T)cell system targeting vascular endothelial growth factor receptor 2(VEGFR2)and to verify its antitumor and regulatory activity in vitro.Methods After verifying and screening the successfully constructed cells,the targeting binding ability of CAR-T cells was verified in vitro.The concentrations of interleukin-2 and interferon-γsecretion from CAR-T cells were detect by kits in vitro,and antitumor activity of CAR-T cells was evaluated by lactate dehydrogenase kit.Continuous variables were expressed as mean±SD,and comparison between groups were performed by t test or analysis of variance.Results Compared with the control groups,the CAR+-T cell+MCA38VEGFR2+group had the strongest target cell killing ability.In the smgCAR-T cell+MCA38VEGFR2+group,the target cell killing rate gradually increased with the increase of Rapalog.The maximum killing rate was(66.25±13.20)%when Rapalog was 40 nmol/L.At 24 h,the killing rate of the two groups increased simultaneously with not statistically significant after Rapalog was added to the smgCAR-T+MCA38VEGFR2+groups[(44.25±6.24)%vs.(49.25±5.56)%,t=1.197,P>0.05].At 36 h,the killing rate of group A(adding Rapalog again)and B(without adding Rapalog again)was statistically significant[(89.00±3.16)%vs.(56.50±6.61)%,t=8.873,P<0.01].Conclusion The smgCAR-T cells exhibited certain antitumor activity in vitro,and its activity was regulated by the small molecule compound Rapalog.

关 键 词：嵌合抗原受体T细胞 血管内皮生长因子受体2 结直肠癌 

分 类 号：R735.34[医药卫生—肿瘤]