Construction of latent membrane protein 2A chimeric antigen receptor-T cells and their lethal effects on nasopharyngeal carcinoma cells / 中华耳鼻咽喉头颈外科杂志
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
; (12): 925-930, 2018.
Artículo
en Chino
| WPRIM (Pacífico Occidental)
| ID: wpr-807765
Biblioteca responsable:
WPRO
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.
Texto completo:
Disponible
Base de datos:
WPRIM (Pacífico Occidental)
Idioma:
Chino
Revista:
Chinese Journal of Otorhinolaryngology Head and Neck Surgery
Año:
2018
Tipo del documento:
Artículo