CD19 CAR基因转染的Nalm-6细胞免疫表型特征分析  

Immunophenotypic characteristics of CD19 chimeric antigen receptor(CAR)-transfected Nalm-6 cells

作　　者：袁婷 刘美静 邓琦[1] 李新[1] 穆娟[1] 江嫣雨 李玉明[1] Yuan Ting;Liu Meijing;Deng Qi;Li Xin;Mu Juan;Jiang Yanyu;Li Yuming(Department of Hematology, Tianjin First Central Hospital, Tianjin 300192, China;The First Central Clinical College of Tianjin Medical University, Tianjin 300019, China)

机构地区：[1]天津市第一中心医院血液科,300192 [2]天津医科大学一中心临床学院,300019

出　　处：《中华微生物学和免疫学杂志》2019年第8期613-619,共7页Chinese Journal of Microbiology and Immunology

摘　　要：目的探讨CAR-T细胞制备过程中,可能出现的CD19 CAR转染入白血病细胞的免疫表型特征。方法倒置显微镜观察Nalm-6细胞CD19 CAR转染后细胞形态变化;流式细胞仪检测CD19 CAR的转染率和CD19 CAR Nalm-6细胞的免疫表型;化学发光法检测培养体系的细胞因子分泌。结果CD19 CAR Nalm-6转染率为(46.50±3.78)%,CD19 CAR KG1a转染率为(15.70±1.22)%;CD19 CAR Nalm-6细胞较Nalm-6细胞增殖迅速(第0天、第4天、第7天、第12天的P值分别为:P0=6.339、P4=3.447、P7=0.012、P12=0.009);CD19 CAR Nalm-6细胞培养过程中出现细胞聚集、互相黏连现象,并逐渐聚集成团;转染率为(46.50±3.78)%的CD19 CAR Nalm-6培养体系CD19表达仅为1.19%,培养体系增加Nalm-6细胞比例后混合培养,CD19表达逐渐增高,而CD22表达无变化;用CD19 CAR Nalm-6细胞培养体系的上清液培养Nalm-6细胞,其CD19表达逐渐下降;CD19 CAR Nalm-6细胞IL-10、TNF-α分泌水平高于Nalm-6细胞。结论通过对CD19 CAR基因被引入白血病细胞中的免疫表型分析,提示我们CD22 CAR-T治疗可以作为CD19 CAR基因转染入白血病细胞的挽救或联合治疗措施。Objective To investigate the immunophenotypic characteristics of potential leukemia cells transfected with CD19 antigen receptor(CAR) during CAR-T cell preparation. Methods Morphological changes in CD19 CAR-transfected cells were observed under inverted microscope. The transfection rate and immunophenotype of transfected Nalm-6 cells were analyzed by flow cytometry. Secretion of cytokines in the culture system was detected by chemiluminescence. Results The transfection rate of Nalm-6 cells by CD19 CAR was (46.50±3.78)% and that of KG1a cells was (15.70±1.22)%. CD19 CAR-transfected Nalm-6 cells proliferated more rapidly than Nalm-6 cells (P values on 0 d, 4 d, 7 d and 12 d were 6.339, 3.447, 0.012 and 0.009). In the culture of CD19 CAR-transfected Nalm-6 cells, cell aggregation and adhesion were observed and they gradually gathered into a group. The rate of CD19 expression was only 1.19% in the CD19 CAR-transfected Nalm-6 cell culture system with the transfection rate of (46.50±3.78)%. After increasing the proportion of Nalm-6 cells in the culture system, CD19 expression was gradually increased, while the expression of CD22 remained stable. CD19 expressed by Nalm-6 cells cultured in the supernatant of CD19 CAR-transfected Nalm-6 cell culture system was decreased gradually. The levels of IL-10 and TNF-α secreted by CD19 CAR-transfected Nalm-6 cells were higher than those by Nalm-6 cells. Conclusions Results of the immunophenotypic analysis of CD19 CAR-transfected leukemia cells suggested that CD22 CAR-T cell therapy could be used as a rescue or combination therapy for CD19 CAR transfection into leukemia cells.

关 键 词：CD19 CAR Nalm-6细胞 转染效率 免疫表型 CD22 

分 类 号：R733.71[医药卫生—肿瘤]