不同信号肽对嵌合抗原受体T细胞杀伤作用的影响研究

李帆; 张琴星; 童祥文; 田高辉; 顾力行; 徐瑶

doi:10.19401/j.cnki.1007-3639.2022.02.006

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不同信号肽对嵌合抗原受体T细胞杀伤作用的影响研究

论著 | 更新时间：2025-12-31

- 不同信号肽对嵌合抗原受体T细胞杀伤作用的影响研究
- A study on influence of different signal peptides on anti-tumor effect of chimeric antigen receptor (CAR) T cells
- 中国癌症杂志 2022年32卷第2期页码：142-151
- 作者机构：
  
  武汉科技大学生命科学与健康学院生物医学研究院,湖北武汉430081
- 作者简介：
  
  徐瑶　E-mail: xuyao0307@wust.edu.cn
- 基金信息：
  
  湖北省技术创新专项(2019ACA168)
- DOI：10.19401/j.cnki.1007-3639.2022.02.006
  中图分类号： R730.51
- 收稿：2021-10-09，
  
  纸质出版：2022-02-28
- 稿件说明：
移动端阅览
李帆, 张琴星, 童祥文, 等. 不同信号肽对嵌合抗原受体T细胞杀伤作用的影响研究[J]. 中国癌症杂志, 2022,32(2):142-151.

Fan LI, Qinxing ZHANG, Xiangwen TONG, et al. A study on influence of different signal peptides on anti-tumor effect of chimeric antigen receptor (CAR) T cells[J]. China Oncology, 2022, 32(2): 142-151.
李帆, 张琴星, 童祥文, 等. 不同信号肽对嵌合抗原受体T细胞杀伤作用的影响研究[J]. 中国癌症杂志, 2022,32(2):142-151. DOI： 10.19401/j.cnki.1007-3639.2022.02.006.

Fan LI, Qinxing ZHANG, Xiangwen TONG, et al. A study on influence of different signal peptides on anti-tumor effect of chimeric antigen receptor (CAR) T cells[J]. China Oncology, 2022, 32(2): 142-151. DOI： 10.19401/j.cnki.1007-3639.2022.02.006.

摘要

背景与目的：

信号肽（signal peptide

SP）是一段存在于前体蛋白N-端的短肽链

能够调节前体蛋白的折叠和转移

在蛋白质的分泌过程中扮演着极其重要的角色。近年来

靶向CD19的嵌合抗原受体（chimeric antigen receptor

CAR）T细胞在白血病治疗中取得了重大突破

关于CAR结构的胞内域改造方面也有诸多研究

而对单链可变片段（scFv）的N端SP研究进展缓慢。探讨4种不同SP的CD19-CAR在T细胞表面表达及对CD19

靶细胞的杀伤作用。

方法：

通过基因合成和分子克隆技术

构建含4种不同SP（SP1、SP2、SP3、SP4）的靶向CD19抗原的CAR载体

进行慢病毒包装

将得到的慢病毒转染T细胞

利用流式细胞术检测细胞转染效率

采用钙黄绿素释放法检测该细胞对靶细胞的杀伤作用

采用酶联免疫吸附实验（enzyme-linked immunosorbent assay

ELISA）检测细胞因子IFN-

#x003b3;和TNF-

#x003b1;的分泌水平。

结果：

成功构建4种不同SP的重组慢病毒载体

将4种慢病毒转导T细胞后

结果显示

分别有20.9%、22.6%、31.5%、38.6%的T细胞表面能够表达CD19-CAR（分别命名为SP1-CD19、SP2-CD19、SP3-CD19和SP4-CD19细胞）

进一步杀瘤实验证明

SP4-CD19细胞对CD19

肿瘤细胞的杀伤作用显著高于SP1-CD19、SP2-CD19和SP3-CD19细胞（

0.01）

并且当效靶比为10

#x02236;1 共培养24 h 后

与SP1-CD19、SP2-CD19和 SP3-CD19细胞相比

SP4-CD19细胞的IFN-

#x003b3;和TNF-

#x003b1;的分泌水平显著升高（

0.05）。此外

4种不同SP的CAR-T对CD19

–

肿瘤细胞K562的杀伤作用差异无统计学意义（

0.05）。

结论：

SP4-CD19细胞的转染效率、细胞因子分泌水平及对CD19

肿瘤细胞的杀伤作用均显著高于SP1-CD19、SP2-CD19和SP3-CD19细胞

该研究成果为CAR-T优化改造及其高效的临床应用奠定了科学基础。

Abstract

Background and purpose:

Signal peptide (SP) is a short peptide chain at the N-terminal of precursor protein

which can regulate the folding and transfer of precursor protein and plays an important role in protein secretion. In recent years

significant breakthroughs have been made in the treatment of leukemia with CD19-targeted chimeric a

ntigen receptor (CAR) T cells

and many achievements have been reported in the intracellular domain modification of CAR structure

while the N-terminal SP of scFv presents less progress. The purpose of this study was to investigate the CD19-CAR expression of four different SP on the surface of T cells and their effect on the killing of CD19

target cells.

Methods:

The CAR vectors containing four different SP (SP1

SP2

SP3

SP4) targeting CD19 antigen were constructed by gene synthesis and molecular cloning technology

and then packaged into lentivirus. The obtained lentivirus was transfected into T cells. The transfection efficiency of the cells was detected by flow cytometry

the killing effect of the cells on target cells was detected by calcein release assay

and the secretion levels of IFN-

#x003b3; and TNF-

#x003b1; were detected by ELISA.

Results:

The recombinant lentiviral plasmids with four different SP were successfully constructed

and the four packaged lentiviruses were transduced into T cells. The results showed that 20.76%

22.29%

31.57% and 38.42% of T cells expressed CD19-CAR (named as SP1-CD19

SP2-CD19

SP3-CD19 and SP4-CD19 cells

respectively)

respectively. The killing effect of SP4-CD19 on CD19

tumor cells was significantly higher compared with SP1-CD19

SP2-CD19 and SP3-CD19 cells (

0.01)

and the secretion levels of IFN-

#x003b3; and TNF-

#x003b1; in SP4-CD19 cells were significantly higher than those in SP1-CD19

SP2-CD19 and SP3-CD19 cells when the effect-target ratio was 10 to 1 for 24 h (

0.05). There was no significant difference in the killing effect of CAR-T on CD19 negative cells K562 among four different SP (

0.05).

Conclusion:

The transfection efficiency and killing effect of SP4-CD19 cells on CD19

tumor cells were significantly higher compared with SP1-CD19

SP2-CD19 and SP3-CD19 cells

which laid a scientific foundation for th

e optimization and efficient clinical application of CAR-T.

关键词

Keywords

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