抗PDL1-CAR慢病毒载体的构建及其转染培养体系的优化

Construction of Anti-PDL1-CAR Lentivirus Vectors and the Optimization of Their Transfection and CultureS ystem

目的:设计构建抗PDL1-CAR慢病毒载体,分析嵌合抗原受体T(chimeric antigen recptor T, CART)细胞的结构和功能,同时优化CART细胞的转染和培养,增加CART细胞的表达率和杀伤活性。方法:构建靶向程序性死亡配体1(programmed death-ligand1,PDL1)的CAR慢病毒载体,按病毒感染复数(multiplicity of infection, MOI)=10、20、40的条件进行转染,采用非磁珠法进行培养扩增,荧光显微镜和流式细胞仪检测转染后CART细胞的表达,最后采用流式绝对计数法检测CART细胞的杀伤性。结果:采用GV401质粒构建的抗PDL1-CAR慢病毒载体,经培养扩增后3组MOI的CART细胞阳性表达率分别为28.80%±0.46%、54.77%±0.32%、71.37%±0.42%,并在荧光显微镜下观察到抗PDL1-CAR细胞表达绿色荧光。在效靶比为2:1、5:1和10:1时,抗PDL1-CAR细胞比T细胞对PDL1-CA46细胞的杀伤率更高(Z=-1.964, P <0.05)。结论:构建抗PDL1-CAR慢病毒载体,可以为其他各种CART细胞的构建提供良好参考,CART细胞的表达率和杀伤活性的提高,为CART细胞的功能研究奠定基础。

Objective:To construct anti-PDL1-CAR lentivirus vectors,analyze the structures and functions of chimeric antigen receptor T(CART)cells,optimize their transfection and culture system,and promote their expressions and cytotoxicity.Methods:CAR lentivirus vectors were constructed to target PD-L1.Vectors were transfected under the conditions of multiplicity of infection(MOI)=10,20 and 40.The cells were amplified by using the non-dynabeads method.Then the expression of CART cells was assessed by a fluorescence microscope and a flow cytometer.Finally,the cytotoxicity of CART cells was quantified based on flow cytometry.Results:Anti-PDL-CAR lentivirus vector was constructed with GV401 plasmid.The expression levels of CART cells were 28.80%±0.46%,54.77%±0.32%and 71.37%±0.42%,respectively.Anti-PDL1-CART cells were in green fluorescence under a fluorescence microscope.The killing activity of anti-PDL1-CART cells was higher than that of T cells to PDL1-CA46 cells in the ratios of 2:1,5:1 and 10:1(Z=-1.964,P<0.05).Conclusion:The construction of anti-PDL1-CAR lentivirus vector can be promising reference for other CART design,and the improvement in the expression and killing activity of CART cells will provide a basis for the investigation of functionsof CART cells.