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目的:通过慢病毒介导获得表达针对血管内皮细胞生长因子受体1(vascular endothelial growth factor receptor 1,VEGFR1/FLT1)的嵌合抗原受体(chimeric antigen receptor,CAR)的Jurkat细胞,探讨其对VEGF的趋向性。方法:合成针对VEGFR1的CAR(FLT1-CAR),构建重组慢病毒载体LV-gn-FLT1-CAR,并感染Jurkat细胞;经G418筛选得到稳定转染细胞株;PCR及流式细胞术检测细胞株中FLT1-CAR mRNA及蛋白的表达,Transwell法检测细胞株对VEGF的趋化效果。结果:重组慢病毒LV-gn-FLT1-CAR成功构建;FLT1-CAR成功整合到Jurkat细胞中并稳定表达FLT1-CAR蛋白。筛选的细胞株Jurkatgn-FLT1-CAR-1、Jurkat-gn-FLT1-CAR-2对VEGF有显著趋化效果,100 ng/ml VEGF处理后,Jurkat-gn-FLT1-CAR-1细胞趋化数为(62±8)个,显著高于对照组Jurkat趋化细胞数的(18±5)个(P<0.01)。结论:成功筛选到稳定表达FLT1-CAR的Jurkat细胞克隆,其对VEGF有明显的趋向作用。
OBJECTIVE: To obtain Jurkat cells expressing chimeric antigen receptor (CAR) targeting vascular endothelial growth factor receptor 1 (VEGFR1 / FLT1) by lentivirus. The trend of VEGF. METHODS: CAR (FLT1-CAR) against VEGFR1 was synthesized and constructed recombinant lentiviral vector LV-gn-FLT1-CAR and infected Jurkat cells. Stably transfected cell lines were screened by G418. PCR and flow cytometry In FLT1-CAR mRNA and protein expression, Transwell assay of cell lines chemotactic effect of VEGF. Results: The recombinant lentivirus LV-gn-FLT1-CAR was successfully constructed. FLT1-CAR was successfully integrated into Jurkat cells and stably expressed FLT1-CAR protein. The chemotactic effect of Jurkatgn-FLT1-CAR-1 and Jurkat-gn-FLT1-CAR-2 on the cell cycle of Jurkat-gn-FLT1-CAR-1 cells after 100 ng / (62 ± 8), which was significantly higher than that of Jurkat chemotactic cells in the control group (18 ± 5) (P <0.01). CONCLUSION: Jurkat cell clones stably expressing FLT1-CAR were successfully screened and showed a clear trend toward VEGF.