摘要
目的探讨抗CD19嵌合抗原受体(CAR)修饰的T细胞(CAR-T)制备阶段Nalm-6细胞的免疫表型及PD-1 mRNA水平变化。方法将30株急性淋巴细胞白血病(ALL)细胞株Nalm-6细胞分为空白组(不作任何处理)、对照组(转染空白CD19质粒pVAX1)、CD19组(转染CD19真核质粒pVAX1-F),每组各10株。CCK-8细胞增殖实验检测Nalm-6细胞增殖率,倒置显微镜观察Nalm-6细胞转染后细胞形态,流式细胞仪检测Nalm-6细胞免疫表型及PD-L1、PD-1 mRNA水平。结果转染24、36 h时,CD19组Nalm-6增殖率低于对照组与空白组,差异有统计学意义(P<0.05)。空白组Nalm-6细胞间隙分明,排列较稀疏;对照组Nalm-6细胞出现少量聚集,排列较密,有粘连现象;CD19组Nalm-6细胞出现大量聚集,排列紧密,有团状现象。CD19组CD19表达量低于对照组与空白组,差异有统计学意义(P<0.05)。各组CD22表达量比较,差异无统计学意义(P>0.05)。Nalm-6细胞CD19表达随着共培养时间延长增高,而CD22表达无显著变化。CD19组PD-1、PD-L1 mRNA水平低于空白组与对照组,差异有统计学意义(P<0.05)。结论CAR-T细胞制备阶段Nalm-6细胞中,CD19表达上调、PD-1 mRNA水平下降,不能显著抑制肿瘤细胞的增殖能力。
Objective To investigate the immunophenotype and PD-1 mRNA levels of Nalm-6 cells during the preparation of anti-CD19 chimeric antigen receptor(CAR)-modified T cells(CAR-T).Methods Thirty acute lymphoblastic leukemia(ALL)cell lines Nalm-6 were divided into blank group(no treatment),control group(transfected with blank CD19 plasmid pVAX1),and CD19 group(transfected with CD19 eukaryotic plasmid pVAX1-f),with 10 strains in each group.The proliferation rate of Nalm-6 cells was detected by CCK-8 cell proliferation assay.The cell morphology after transfection of Nalm-6 cells was observed by inverted microscope.The immunophenotype of Nalm-6 cells and the mRNA levels of PD-l1 and PD-1 were detected by flow cytometry.Results At 24 and 36 hours after transfection,the proliferation rate of Nalm-6 in the CD19 group was lower than that in the control group and the blank group,and the difference was statistically significant(P<0.05).In the blank group,Nalm-6 cells were well spaced and sparsely arranged.In the control group,Nalm-6 cells showed a small amount of aggregation,dense arrangement and adhesion.The Nalm-6 cells in the CD19 group were clustered in large numbers and arranged closely,with a cluster-like appearance.The expression level of CD19 in CD19 group was lower than that in control group and blank group(P<0.05).There was no significant difference in the expression of CD22 in each group(P>0.05).The expression of CD19 in Nalm-6 cells increased with the extension of co-culture time,while the expression of CD22 did not change significantly.The mRNA levels of PD-1 and PD-l1 in CD19 group were lower than those in blank group and control group(P<0.05).Conclusion In Nalm-6 cells at the stage of CAR-T cell preparation,the expression of CD19 is up-regulated and the level of PD-1 mRNA is reduced,which can not significantly inhibit the proliferation of tumor cells.
作者
彭芸
柏涌海
孙文江
张桂丽
PENG Yun;BAI Yong-hai;SUN Wen-jiang;ZHANG Gui-li(Naval Medical University,Shanghai 200433,China;Department of Prevention and Health Care,Changzheng Hospital,Naval Medical University,Shanghai 200003,China;Department of Rehabilitation Medicine,Shanghai First People's Hospital,Shanghai 200080,China;Department of Surgery,Putuo District Central Hospital,Shanghai 200080,China)
出处
《临床军医杂志》
CAS
2020年第6期661-664,共4页
Clinical Journal of Medical Officers
