摘要
目的建立嵌合抗原受体T(CAR-T)细胞治疗相关细胞因子释放综合征(CRS)的小鼠模型。方法通过分子克隆及慢病毒转染技术,构建靶向人CD19分子的CAR-T细胞,采用流式细胞术检测CAR-T细胞转染效率,酶联免疫吸附试验(ELISA)法及流式细胞术检测CAR-T细胞特异性杀伤靶细胞的能力。通过尾静脉注射CAR-T细胞至荷瘤重症联合免疫缺陷裸鼠体内,小鼠分为磷酸缓冲液组、低负荷组(注射1×105个人淋巴瘤细胞Raji-Luc2细胞)和高负荷组(注射5×105个Raji-Luc2细胞)。采用动物活体成像法检测肿瘤治疗效果,ELISA法检测小鼠血清中细胞因子人白细胞介素2(IL-2)、人γ-干扰素(IFN-γ)、鼠IL-6、鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF)的水平。结果经尾静脉注射人T细胞和T细胞+OKT-3抗体后,T细胞组和T细胞+OKT-3组小鼠的健康得分分别为(1.15±0.08)分和(2.90±0.15)分,差异有统计学意义(P<0.001)。T细胞+OKT-3组小鼠血清中人IL-2、人IFN-γ、人IL-15、鼠IL-6、鼠GM-CSF水平分别为(1064.00±50.14)、(1285.00±193.90)、(202.4±18.76)、(1478.00±289.20)和(350.70±42.27)pg/ml,均高于T细胞组[分别为(22.67±6.36)、(23.67±3.71)、(44.33±14.45)、(147.30±36.20)和(138.00±22.74)pg/ml,均P<0.05];OKT-3联合人T细胞引起小鼠血清中细胞因子人IL-2、人IFN-γ、鼠IL-6、鼠GM-CSF水平迅速升高,并伴有体温升高及体重下降。成功构建靶向CD19分子的CAR-T细胞,流式细胞术检测CAR-T细胞阳性率>30%。ELISA结果显示,在CD19抗原存在时,CAR-T19细胞与Raji、Nalm-6共孵育组细胞分泌IL-2和IFN-γ的水平分别为(561.00±37.07)、(680.30±71.27)、(369.00±25.71)和(523.00±26.31)pg/ml,均高于与K562共孵育组[分别为(55.00±20.53)和(64.00±7.55)pg/ml,均P<0.001]。小鼠成像实验显示,小鼠成瘤后第7天经尾静脉回输活化后的CAR-T19细胞,成瘤第13天,低负荷和高负荷组小鼠肿瘤荧光强度均低于接种肿瘤的第7天,高负荷组肿瘤荧光
Objective To establish a cytokine release syndrome(CRS)mouse model related to CAR-T cell therapy and provide a research model for the clinical phenomena.Methods CAR-T cells targeting human CD19 molecule were constructed by molecular cloning and lentiviral transfection.Flow cytometry(FACS)was used to detect the transfection efficiency of CAR-T cells.The tumor-killing efficiency of CAR-T cells was detected by ELISA and flow cytometry.The CAR-T cells were injected into the tumor-bearing SCID/Beige mice through tail vein,and divided into phosphate buffered solution(PBS)group,low-burden group(1×105 Raji-Luc2 cells)and high-burden group(5×105 Raji-Luc2 cells).The tumor treatment effect was detected by animal in vivo imaging.Serum levels of cytokines including human IFN-γ,human IL-2,mouse IL-6,and mouse GM-CSF were measured by ELISA.The health status of the mice was evaluated by pathological examination.Results The health scores of T cell group and T cell+OKT-3 group were(1.15±0.08)and(2.90±0.15),respectively,after the injection of human T cell and T cell+OKT-3 antibody through tail vein,and the difference was statistically significant(P<0.001).The serum levels of human IL-2,human IFN-γ,human IL-15,mouse IL-6 and mouse GM-CSF in T cell+OKT-3 group were(1064.00±50.14),(1285.00±193.90),(202.4±18.76),(1478.00±289.20)and(350.70±42.27)pg/ml,respectively,higher than(22.67±6.36),(23.67±3.71),(44.33±14.45),(147.30±36.20),(138.00±22.74)pg/ml in T cell group(P<0.05).OKT-3 combined with human T cells caused a rapid increase in serum levels of human IL-2,human IFN-γ,mouse IL-6 and mouse GM-CSF,accompanied by an increase in body temperature and weight loss.CD19-targeting CAR-T cells were successfully constructed,and the positive rate of CAR-T cells was>30%detected by flow cytometry.ELISA results showed that in the presence of CD19 antigen,IL-2 and IFN-γsecreted by CAR-T19 cells co-incubated with Raji and Nalm were(561.00±37.07),(680.30±71.27),(369±25.71)and(523.00±26.31)pg/ml,respectively,higher than(55.00±20.
作者
单吉琦
南书锋
李峰
申春一
张毅
Shan Jiqi;Nan Shufeng;Li Feng;Shen Chunyi;Zhang Yi(Biotherapy Center,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2021年第12期1248-1254,共7页
Chinese Journal of Oncology
基金
国家自然科学基金面上项目(81771781)。
