CD19-CAR-T和CD19-CAR-CIK细胞对表达CD19+K562细胞株杀伤作用的比较  

Comparing the cytotoxicity and specificity of CAR-19-CIK and CAR-19-T effector cells in vitro against K562 cell line expressing CD19

作　　者：曹韪凡 羊轶驹[2] 康立清[3] 余宙 赵世民 俞磊 曹峰林 戴绍军[1] 

机构地区：[1]东北林业大学 [2]海南省三亚农垦医院 [3]上海市华东师范大学化学与分子工程学院 [4]上海分子治疗与新药创制工程技术研究中心 [5]生物医学工程与技术研究所 [6]哈医大附属第一医院

出　　处：《中国优生与遗传杂志》2017年第6期28-31,50,共5页Chinese Journal of Birth Health & Heredity

基　　金：海南省医药卫生科研课题支持;课题编号:1321320.24A1005

摘　　要：目的 CAR-CD19-CIK和CAR-CD19-T效应细胞在体外对表达CD19的K562稳定细胞株杀伤能力和杀伤特异性的比较。方法效应细胞的构建是以通过基因合成和分子克隆手段构建CAR-CD19片段,以酶切的方法将CAR-CD19片段插入到慢病毒载体上;分离获得同一个健康志愿者外周血单核细胞,一部分加IFN-γ、重组人IL-1α、CD3单克隆抗体、重组人IL-2等细胞因子诱导CIK细胞产生;另一部分体外分选CD3阳性T细胞,CAR-19慢病毒同时感染CIK细胞和T细胞,采用流式细胞术检测转导效率和细胞分型;CD19+的靶细胞是由装载CD19基因表达的慢病毒载体转导K562细胞系,通过抗菌素的压力选择而构建;乳酸脱氢酶释放法(LDH)检测CAR-CIK细胞和CAR-T细胞对靶细胞的杀伤效率,炎性因子检测(CBA)法检测由CAR-CIK细胞和CAR-T细胞的细胞因子的分泌水平。结果同一CD19-CAR慢病毒载体在相同转导条件下,对原代CIK细胞的转导效率是60.5%,对原代T细胞的转导效率是36.2%;当效靶比5:1、10:1和20:1在这三种比例时,CAR-19-CIK细胞对CD19-K562细胞的杀伤效率分别为14.65%、33.08%和42.5%,对K562细胞的杀伤效率分别为8.3%、10.1%和24.9%;而CAR-19-T细胞对CD19-K562细胞的杀伤效率分别为17.22%、26.52%和32.49%,对k562细胞的杀伤效率分别为0.1%、3.6%和10.3%。结论 CAR-19-CIK细胞的杀伤作用明显强于CIK细胞;CAR-19-T细胞杀伤特异性比CAR-19-CIK细胞强。本研究对CAR-T和CAR-CIK免疫细胞治疗血液肿瘤的新策略和新临床方案设计提供了有意义的实验数据。Objective： To compare the cytotoxicity and specificity of CAR-19-CIK and CAR-19-T effector cells in vitro against K562 cell line expressing CD19. Methods： The effector cells （CAR-19-CIK and CAR-19-T） were constructed by delivering the CAR-CD19 gene expression cassette vis a lentiviral vector that carried the CAR-CD19 fusion gene to CIK and CD3＋ T cells that provided by a same healthy volunteerwith different isolation and culture method. The transduction efficiency and cell typing of CAR-19 lentivirustransduced CIK cells and T cells were analyzed by flow cytometry cells. The target cell CD19- K562stable cell line was prepared by lentiviral vector transduction of a CD19 gene and through the selective pressure of antibiotics. Lactate dehydrogenase release assay （LDH） was used to detect the cytotoxicity of CAR-19-CIK cells and CAR-19-T cells to target cells, and the levels of cytokines expressed and secreted by CAR-CIK cells and CAR-T cells were detected by inflammatory factor assay （CBA） . Results： The transduction efficiency of the CIKcellswere60.5%, and the transduction efficiency of the primary T cells were about 36.2%. The effector-target ratio was set as 5： 1, 10：1 and 20： 1, killing efficiency of CAR-19- CIK cells to CD19-K562 cells were 14.65%, 33.08% and 42.5%, while on the cytotoxicity of K562 cells, negative target cells, were 8.3%, 10.1% and 24.9%. and the killing efficiency of CAR-19 -T cells to CD19-K562 cells were 17.22%, 26.52% and 32.49%, while to K562 cells, the killing efficiency were 0.1%, 3.6% and 10.3%. Conclusion： the cytolytic effect of CAR-19- CIK cells was stronger than that of CIK cells without expression of CD19 targeting marker; and the specificity of CAR-19 -T cells was stronger than that of CAR-19-CIK cells. The present study provides valuable experimental data for the design of new strategies and new clinical protocols for CAR-T and CAR-CIK immune cells in the treatment of hematologic malignancies.

关 键 词：过继性免疫疗法 嵌合抗原受体 CAR-T CAR-CIK 

分 类 号：R730.51[医药卫生—肿瘤]