钠碘转运体报告基因共表达对嵌合型受体T细胞体外增殖及杀伤能力的影响  

Effects of sodium iodide symporter co-expression on proliferation and cytotoxic activity of chimeric antigen receptor T cells in vitro

作　　者：田川卉子 黄普凤 何雨佳 王亮 彭志平 TIAN Chuanhuizi;HUANG Pufeng;HE Yujia;WANG Liang;PENG Zhiping(Department of Radiation Medicine,College of Basic Medicine,Chongqing Medical University,Chongqing 400014,China)

机构地区：[1]重庆医科大学基础医学院放射医学教研室,重庆400014

出　　处：《南方医科大学学报》2022年第7期1062-1068,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(81771871)。

摘　　要：目的探讨钠碘转运体(NIS)报告基因的共表达对嵌合型受体T(CAR-T)细胞体外增殖和杀伤活性的影响。方法慢病毒感染法制备CAR-T细胞(仅表达CD19 CAR的T细胞)、NIS-T细胞(仅表达NIS报告基因的T细胞)和NIS-CAR-T细胞(共表达NIS和CD19 CAR的T细胞),然后按T细胞蛋白表达情况分为4组∶T细胞组(未转染的T细胞)、CAR-T组、NIS-T组、NIS-CAR-T组。流式细胞术检测各组NIS和CAR转染率。各组细胞常规培养24、48、72 h,CCK-8法检测增殖能力。各组T细胞为效应细胞,Nalm6肿瘤细胞为靶细胞,按效靶比0.5∶1、1∶1、2∶1、4∶1共培养24、48、72 h,乳酸脱氢酶细胞毒性检测法(LDH)检测各组细胞对肿瘤细胞的杀伤率,酶联免疫吸附试验(ELISA)法检测共培养上清液中各组细胞因子人干扰素-γ(IFN-γ)和人肿瘤坏死因子-β(TNF-β)释放水平。摄碘实验检测表达NIS蛋白各组细胞的NIS功能。结果CAR-T细胞、NIS-CAR-T细胞的CAR转染率分别为91.91%、99.41%,NIS-T细胞、NIS-CAR-T细胞的NIS转染率分别为47.83%、50.24%。常规培养24、48、72 h CAR-T细胞与NIS-CAR-T细胞增殖率差异无统计学意义(P>0.05)。0.5∶1、1∶1、2∶1和4∶1效靶比作用24 h时CAR-T细胞杀伤率(%)均高于NIS-CAR-T细胞(P<0.05),作用48 h和72 h时两组杀伤率差异均无统计学意义(P>0.05)。CAR-T细胞与NIS-CAR-T细胞均存在IFN-γ和TNF-β释放现象,释放水平均高于对照组(P<0.05)。NIS-T细胞与NIS-CAR-T细胞均具有特异性摄碘能力,二者间摄碘能力差异无统计学意义(P>0.05),但均高于对照组T细胞(P<0.05)。结论NIS报告基因的共表达不影响CAR-T细胞中CAR转染率,对细胞增殖和杀伤活性无抑制现象。Objective To investigate the effects of co-expression of sodium iodide symporter(NIS)reporter gene on the proliferation and cytotoxic activity of chimeric antigen receptor(CAR)-T cells in vitro.Methods T cells expressing CD19 CAR(CAR-T cells),NIS reporter gene(NIS-T cells),and both(NIS-CAR-T cells)were prepared by lentiviral infection.The transfection rates of NIS and CAR were determined by flow cytometry,and the cell proliferation rate was assessed using CCK-8 assay at 24,48 and 72 h of routine cell culture.The T cells were co-cultured with Nalm6 tumor cells at the effector-target ratios of 1∶2,1∶1,2∶1 and 4∶1 for 24,48 and 72 h,and the cytotoxicity of CAR-T cells to the tumor cells was evaluated using lactate dehydrogenase(LDH)assay.ELISA was used to detect the release of IFN-γand TNF-βin the co-culture supernatant,and the function of NIS was detected with iodine uptake test.Results The CAR transfection rate was 91.91%in CAR-T cells and 99.41%in NIS-CAR-T cells;the NIS transfection rate was 47.83%in NIS-T cells and 50.24%in NIS-CAR-T cells.No significant difference in the proliferation rate was observed between CAR-T and NIS-CAR-T cells cultured for 24,48 or 72 h(P>0.05).In the co-cultures with different effector-target ratios,the tumor cell killing rate was significantly higher in CAR-T group than in NIS-CAR-T group at 24 h(P<0.05),but no significant difference was observed between the two groups at 48 h or 72 h(P>0.05).Higher IFN-γand TNF-βrelease levels were detected in both CAR-T and NIS-CAR-T groups than in the control group(P<0.05).NIS-T cells and NIS-CAR-T cells showed similar capacity of specific iodine uptake(P>0.05),which was significantly higher than that in the control T cells(P<0.05).Conclusion The co-expression of the NIS reporter gene does not affect CAR expression,proliferation or tumor cell-killing ability of CAR-T cells.

关 键 词：CAR-T细胞 钠碘转运体 基因表达 体外增殖 杀伤活性 

分 类 号：R733[医药卫生—肿瘤]