荧光定量PCR检测c-Met CAR病毒感染T细胞的效率

Detection of the efficiency of c-Met CAR virus infected T cells by qRT-PCR

目的:通过设计特异性引物,应用荧光定量PCR方法检测c-Met CAR病毒感染T细胞的效率。方法:应用基因重组技术构建c-Met CAR(GFP)逆转录病毒质粒。应用病毒包装技术制备c-Met CAR病毒与c-Met CAR(GFP)病毒,感染T细胞制备c-Met CAR-T细胞与c-Met CAR-T(GFP)细胞。Western blot检测c-Met CAR和c-Met CAR(GFP)在293T细胞中表达的外源性CD3ζ蛋白。设计特异性引物应用荧光定量PCR检测CAR病毒对T细胞的感染效率,并与流式细胞术的检测结果进行比较。结果:构建c-Met CAR(GFP)质粒,荧光显微镜可观察到c-Met CAR(GFP)质粒在293T细胞上表达绿色荧光蛋白。c-Met CAR和c-Met CAR(GFP)病毒感染的293T细胞可表达外源性CD3ζ蛋白。荧光定量PCR检测c-Met CAR与c-Met CAR(GFP)的感染效率分别为(52.1±1.7)%、(55.9±2.3)%。流式细胞术检测c-Met CAR(GFP)病毒感染效率为(50.7±3.6)%,两种检测方法比较差异无统计学意义(P>0.05)。结论:通过设计特异性引物,应用荧光定量PCR方法能够特异性检测CAR病毒对T细胞的感染效率,该检测方法结果准确、安全,对CAR-T细胞的临床应用具有实用价值。

Objective：To detect the efficiency of c-Met CAR infection in T cells by q RT-PCR using specific primers. Methods：Gene recombination technology was introduced to construct c-Met CAR（GFP）retroviral plasmids. C-Met CAR and c-Met CAR（GFP）viruses were prepared by viral packaging. C-Met CAR-T cells and c-Met CAR-T（GFP）cells were prepared by infection of T cells with virus. The expression of c-Met CAR and c-Met CAR（GFP）virus infected 293 T cells was tested by Western blot assay to express exogenous CD3ζ protein. Specific primers were designed to detect the infection efficiency of T cells by q RT-PCR and compared with flow cytometry. Results：c-Met CAR（GFP）plasmid was constructed,and the expression of c-Met CAR（GFP）plasmid on 293 T cells was observed by fluorescence microscope. The results showed that c-Met CAR and c-Met CAR（GFP）virus infected 293 T cells expressed exogenous CD3ζ protein. The infection efficiency of c-Met CAR and c-Met CAR（GFP）virus were（55.9 ± 2.3）% and（52.1 ±1.7）% by q RT-PCR. The efficiency of c-Met CAR（GFP）infection was（50.7 ± 3.6）% by flow cytometry. There was no statistical difference between the two methods（P〉0.05）. Conclusion：Through the design of specific primers,the q RT-PCR can detect the infection efficiency of c-Met CAR virus on T cells,which is accurate and security,and have value for the clinical application of CAR-T cell therapy.