[ROR1靶向嵌合抗原受体修饰T细胞的构建及其对ROR1阳性肿瘤细胞的杀伤作用]

[The Construction of ROR1 Targeting Chimeric Antigen Receptor Modified T Cells and Its Killing Effect for ROR1-positive Tumor Cells].

作者信息

Chen Yue, Mo Ze-Ming, Qin Di-Yuan, Guo Fu-Chun, Liao Xue-Lian, Hu Min, Chen Qian, Wang Yong-Sheng

机构信息

Department of Cancer Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.

Breast Surgery, Chongqing Medical University, Chongqing 400016, China.

出版信息

Sichuan Da Xue Xue Bao Yi Xue Ban. 2019 Mar;50(2):145-151.

PMID:31106530

Abstract

OBJECTIVE

To test the killing effect of type Ⅰ receptor tyrosine kinase-like orphan receptor (ROR1) chimeric antigen receptor T cell (CAR-T) on several ROR1-expressing tumor cells

METHODS

The CAR gene was designed and synthesized by constructing the lentiviral vector plasmid, and HⅠ/RⅠ was used to identify the plasmid. The expression levels of ROR1 among a variety of tumor cell lines were compared using flow cytometry (FCM). The killing effect of CAR-T on positive cells was detected by FCM, the LDH assay and ELISA.

RESULTS

The double enzyme digestion identified CAR gene was successfully constructed to the lentivirus vector plasmid. FCM detection showed that the efficiency of CAR-T infection was about 47.23%. Multiple tumor cells expressed ROR1 in varying degrees. The FCM and the LDH assay indicated that CAR-T specifically killed ROR1-positive tumor cells. On positive target cells, more interferonI-γ (FN-γ) could be released during the CAR-T killing process than control T (<0.05).

CONCLUSION

We successfully constructed ROR1 CAR-T. CAR-T can specifically kill ROR1-positive tumor cells and cause the release of large amounts of IFN-γ, providing an experimental basis for clinical application.

摘要

目的

检测Ⅰ型受体酪氨酸激酶样孤儿受体（ROR1）嵌合抗原受体T细胞（CAR-T）对几种表达ROR1的肿瘤细胞的杀伤作用

方法

通过构建慢病毒载体质粒设计并合成CAR基因，用HⅠ/RⅠ对质粒进行鉴定。采用流式细胞术（FCM）比较多种肿瘤细胞系中ROR1的表达水平。通过FCM、乳酸脱氢酶（LDH）检测法和酶联免疫吸附测定（ELISA）检测CAR-T对阳性细胞的杀伤作用。

结果

双酶切鉴定表明CAR基因成功构建到慢病毒载体质粒中。FCM检测显示CAR-T感染效率约为47.23%。多种肿瘤细胞不同程度表达ROR1。FCM和LDH检测法表明CAR-T可特异性杀伤ROR1阳性肿瘤细胞。在阳性靶细胞上，CAR-T杀伤过程中释放的干扰素-γ（IFN-γ）比对照T细胞多（<0.05）。