Construction of latent membrane protein 2A chimeric antigen receptor-T cells and their lethal effects on nasopharyngeal carcinoma cells
10.3760/cma.j.issn.1673-0860.2018.12.010
- VernacularTitle: 潜伏膜蛋白2A嵌合抗原受体－T细胞的制备及对鼻咽癌细胞杀伤作用的研究
- Author:
Yuan CHEN
1
;
Renjie CHEN
1
;
Xiaochen HUANG
1
;
Genxiong TANG
1
;
Xingwang KUAI
1
;
Mingjiong ZHANG
1
;
Dawei ZHANG
1
;
Qi TANG
1
;
Jin ZHU
1
;
Zhenqing FENG
1
Author Information
1. Department of Otorhinolaryngology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 200031, China
- Publication Type:Journal Article
- Keywords:
Nasopharyngeal neoplasmas;
Membrane proteins;
Chimeric antigen receptor
- From:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
2018;53(12):925-930
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To produce latent membrane protein 2A (LMP2A) chimeric antigen receptor (CAR)-T cells and detect the lethal effect of LMP2A CAR-T cells on nasopharyngeal carcinoma (NPC) cells.
Methods:The study was conducted from September 2016 to December 2017.Genetic engineering technology was used to construct anti-LMP2A CAR lentiviral expression vector and sequencing was identified. The expression of anti-LMP2A CAR in the 293T cells was confirmed by western blot. CCK8 assay was used to evaluate the cytotoxicity of LMP2A CAR-T cells to NPC cells. ELISA assay was performed to test IL-2 and IFN-γ releasing of activated LMP2A CAR-T cells. The inhibition effect of LMP2A CAR-T cells on NPC xenograft tumor was observed in vivo. Statistical analysis was performed by statistical software SPSS 21.0.
Results:The results of PCR and sequencing showed that anti-LMP2A CAR lentiviral expression vector was constructed successfully. The result of western blot indicated the expression of anti-LMP2A CAR in the 293T cells effectively. The results of CCK-8 assay showed that the killing activities of LMP2A CAR-T cells to LV-LMP2A-CNE1 cells were (72.11±9.75)%, (54.65 ±5.42)% and (36.68±3.80)% at 20∶1, 10∶1 and 5∶1 ratio of effective cells to target cells, and had a statistical difference compared to CD19 CAR-T cells and T cells (P<0.05). There was no significant difference in the killing activities of LMP2A CAR-T cells to CNE1 cells compared with CD19 CAR-T cells and T cells. The results of ELISA showed that the content of IL-2 and IFN-γ in the co-culture supernatant of LMP2A CAR-T cells and LV-LMP2A-CNE1 cells was significantly higher than that of LMP2A CAR-T cells and CNE1 cells which had statistical difference (P<0.05); In vivo experiment, the volume of LMP2A CAR-T cell group was (80.3±10.0) mm3 which was significantly lower than that of the control groups, and the difference was statistically significant (P<0.05).
Conclusion:LMP2A CAR-T cells are successfully prepared and have an obvious targeting cytotoxicity on LMP2A-positive NPC cells.
