基于颗粒酶B启动子的磁共振报告基因成像监测CAR-T细胞激活状态

倪晓英; 秦勇; 贺小娅; 黄杰; 张湘敏; 祝慧如; 胡倩; 蔡金华

doi:10.16016/j.2097-0927.202312100

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基于颗粒酶B启动子的磁共振报告基因成像监测CAR-T细胞激活状态

基础医学 | 更新时间：2026-01-09

- 基于颗粒酶B启动子的磁共振报告基因成像监测CAR-T细胞激活状态
- Magnetic resonance imaging based on a granzyme B promoter-driven reporter gene expression monitors CAR-T cell activation
- 陆军军医大学学报 2024年46卷第17期页码：1959-1968
- 作者机构：
  
  1.400014 重庆, 重庆医科大学附属儿童医院, 国家儿童健康与疾病临床医学研究中心, 儿童发育疾病研究教育部重点实验室：放射科
  2.400014 重庆, 重庆医科大学附属儿童医院, 国家儿童健康与疾病临床医学研究中心, 儿童发育疾病研究教育部重点实验室：儿童神经发育与认知障碍重庆市重点实验室
- 作者简介：
  
  [蔡金华，E-mail：cai_jinhua@126.com CAI Jinhua, E-mail: cai_jinhua@126.com
- 基金信息：
  
  中国博士后科学基金第74批面上资助(2023M740456);重庆市自然科学基金面上项目(cstc2020jcyj-msxmX0235)
- DOI：10.16016/j.2097-0927.202312100
  中图分类号： R392.12;R445.2;R73-362
- 收稿：2023-12-19，
  
  修回：2024-02-28，
  
  纸质出版：2024-09-15
- 稿件说明：
移动端阅览
倪晓英, 秦勇, 贺小娅, 等. 基于颗粒酶B启动子的磁共振报告基因成像监测CAR-T细胞激活状态[J]. 陆军军医大学学报, 2024,46(17):1959-1968.

Xiaoying NI, Yong QIN, Xiaoya HE, et al. Magnetic resonance imaging based on a granzyme B promoter-driven reporter gene expression monitors CAR-T cell activation[J]. Journal of Army Medical University, 2024, 46(17): 1959-1968.
倪晓英, 秦勇, 贺小娅, 等. 基于颗粒酶B启动子的磁共振报告基因成像监测CAR-T细胞激活状态[J]. 陆军军医大学学报, 2024,46(17):1959-1968. DOI： 10.16016/j.2097-0927.202312100.

Xiaoying NI, Yong QIN, Xiaoya HE, et al. Magnetic resonance imaging based on a granzyme B promoter-driven reporter gene expression monitors CAR-T cell activation[J]. Journal of Army Medical University, 2024, 46(17): 1959-1968. DOI： 10.16016/j.2097-0927.202312100.

摘要

目的

利用颗粒酶B(granzyme B

GB)启动子控制铁蛋白(ferritin heavy chain

FTH1)报告基因表达，探讨通过磁共振成像(magnetic resonance imaging

MRI)监测嵌合抗原受体T细胞(chimeric antigen receptor T cells

CAR-T细胞)激活状态的可行性。

方法

通过Ficoll密度梯度离心法以及流式分选得到细胞毒性T淋巴细胞(cytotoxic T lymphocytes

CTLs)。将GB启动子和FTH1基因连接

连同二唾液酸神经节苷脂(disialoganglioside 2

GD2)嵌合抗原受体(chimeric antigen receptor

CAR)以慢病毒为载体转入CTLs

构建GD2-CAR-T/pGB-FTH1细胞

以GD2-CAR-T/pCMV-FTH1、GD2-CAR-T和T细胞为对照。CytoTox96@非放射性细胞毒性检测各组细胞与人神经母细胞瘤细胞(SK-N-SH)共培养后的杀伤效果，ELISA检测共孵育因子以及GB分泌量，Western blot、普鲁士蓝染色、细胞MRI检测共培养后FTH1基因的表达。

结果

成功获得CTLs并构建GD2-CAR-T/pGB-FTH1、GD2-CAR-T/pCMV-FTH1、GD2-CAR-T细胞

3组细胞对肿瘤细胞杀伤效果、共孵育因子以及GB分泌量均显著高于T细胞组，GB表达水平在与SK-N-SH细胞共培养后1 d最高

3 d和7 d依次降低。GD2-CAR-T/pGB-FTH1组FTH1相对表达量以及铁含量变化趋势与GB表达一致

MRI信号呈逐渐升高趋势。GD2-CAR-T/pCMV-FTH1组FTH1相对表达量、铁含量及MRI信号在各时间点均无显著差异。GD2-CAR-T和T细胞组无明显FTH1表达及聚铁效应。

结论

基于GB启动子的MRI报告基因成像可实时反映CAR-T细胞的GB表达水平和肿瘤杀伤作用

为CAR-T治疗提供了一种监测细胞激活状态的可视化手段。

Abstract

Objective

To investigate the feasibility of granzyme B (GB) promoter-controlled ferritin heavy chain (FTH1) reporter gene expression for monitoring the activation status of chimeric antigen receptor T cells (CAR-T) by magnetic resonance imaging (MRI).

Methods

Cytotoxic T lymphocytes (CTLs) were screened by Ficoll density gradient centrifugation and flow sorting. The GB promoter and FTH1 gene were ligated together with disialoganglioside 2 (GD2) CAR

and lentiviral vectors were transfected into CTLs to construct GD2-CAR-T/pGB-FTH1 cells. GD2-CAR-T/pCMV-FTH1

GD2-CAR-T

and T cells served as control cells. CytoTox96@non-radioactive cytotoxicity was used to detect the killing effect of each group of cells after co-culture with human neuroblastoma cells (SK-N-SH). ELISA was employed to detect the coincubation factor as well as the amount of GB secretion. Western blotting

Prussian blue staining and cellular MRI were applied to detect the expression of the FTH1 gene after co-culture.

Results

CTLs were successfully obtained

and then GD2-CAR-T/pGB-FTH1

GD2-CAR-T/pCMV-FTH1 and GD2-CAR-T cells were constructed. The killing effect

co-incubation factor and GB secretion of the above 3 groups of cells were significantly higher than those of the T cells

and the level of GB expression was highest at day 1

and then decreased in order at day 3 and day 7 after co-culturing with SK-N-SH cells. The relative expression of FTH1 and iron content of the GD2-CAR-T/pGB-FTH1 cells showed the same trend as GB expression

and the MRI signals were gradually increased. There were no significant differences in the relative expression of FTH1

iron content and MRI signals in the GD2-CAR-T/pCMV-FTH1 cells at all time points. No FTH1 expression or iron aggregation was observed in the GD2-CAR-T and T cells groups.

Conclusion

MRI based on the FTH1 reporter gene driven by the granzyme B promoter can reflect the GB expression level and tumor killing effect of CAR-T cells

which provides a potential real-time visual means to monitor the cell activation status for CAR-T therapy.

关键词

Keywords

references

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