南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (7): 1062-1068.

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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

田川卉子，黄普凤，何雨佳，王 亮，彭志平   

  1. 重庆医科大学基础医学院放射医学教研室，重庆 400014
  • 出版日期:2022-07-20 发布日期:2022-07-15

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

TIAN Chuanhuizi, HUANG Pufeng, HE Yujia, WANG Liang, PENG Zhiping   

  1. Department of Radiation Medicine, College of Basic Medicine, Chongqing Medical University, Chongqing 400014, China
  • Online:2022-07-20 Published:2022-07-15

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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-CART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。

关键词: CAR-T细胞；钠碘转运体；基因表达；体外增殖；杀伤活性

Abstract: 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-γ and TNF-β in 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-γ and TNF-β release 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.

Key words: chimeric antigen receptor T cells; sodium iodide symporter; co-expression; proliferation in vitro; killing activity