Objective: To improve the MigR1-CD19-CAR (chimeric antigen receptor) that contains a single chain variable region (scFv) which targeted to CD19 through a retroviral vector transduction efficiency of T-lymphocytes.
Methods: Insert the CD19-CAR fragment into the retroviral vector (MigR1) through recombinant DNA technology, after transfecting plat-A packaging cell lines, viral supernatant was collected to transduce K562 cell line and activated human T-lymphocytes. We used flow cytometry to determine the transduction efficiency and RT-PCR to confirm the transcription of CD19-CAR gene. The ability of the transduced T cells to produce IFN-γ and TNF-α in a CD19-specific manner was measured in an enzyme-linked immunosorbent (ELISA) assay.
Results: (1)Using MigR1-CD19-CAR retroviral vector to produce the high titer retrovirus. (2)MigR1-CD19-CAR transduction efficiency of K562 cell line was significantly higher than human T-lymphocytes (P<0.01). (3)120 min centrifugation could significantly improve transduction efficiency of T-lymphocytes to (54.5±14.6)%. (4)Transduction efficiency could be improved by deciding transduce time according to T-lymphocytes proliferation fold in vitro individually, and the highest transduction efficiency in the study was 69.3%. The CD19-CAR gene sequence was transcripted specificly with high efficiency. (5) IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased to (13 230±1 543) pg/ml and (4 217±211) pg/ml when coculture with CD19-K562 cells.
Conclusion: We have successfully constructed a second generation CAR which targeted to CD19 through a retroviral vector called MigR1 (MigR1-CD19-CAR). Deciding transduce time according to T-lymphocytes proliferation fold in vitro individually and 120 min centrifugation could improve the CAR transduction efficiency of T-lymphocytes. RT-PCR confirmed that the CD19-CAR gene was specificly transcripted with high efficiency. IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased when activated by target cells.
目的: 优化二代CD19嵌合抗原受体（CD19-CAR）逆转录病毒载体对人原代T细胞的转染效率。
方法: 应用重组DNA技术将CD19-CAR构建入逆转录病毒载体，转入plat-A包装细胞收集病毒上清并转染人原代T细胞及K562细胞系，采用流式细胞术检测不同条件下的转染效率，用RT-PCR方法检测目的基因的表达情况，用ELISA法检测IFN-γ、TNF-α表达水平。
结果: ①MigR1-CD19-CAR逆转录病毒载体成功构建后包装高滴度逆转率病毒；②相同条件下，MigR1-CD19-CAR逆转录病毒对K562细胞系转染效率显著高于T细胞；③对T细胞，离心120 min组的转染率为（54.5±14.6)%，明显高于未离心组、离心30 min组及离心60 min组（P值均<0.05）；④CD3/CD28磁珠与rhIL-2联合对人T细胞活化扩增效率个体差异显著，根据扩增倍数优化个体转染时机可提高转染效率（最高达69.3%)，且RT-PCR检测证实CD19-CAR目的基因在转染后72 h T细胞中高效特异性转录；⑤CD19-CAR-T细胞与CD19-K562细胞共培养组IFN-γ、TNF-α表达水平均增加，分别为（13 230±1 543)pg/ml、(4 217±211)pg/ml，与阴性对照组比较差异均有统计学意义（P值均<0.01）。
结论: 通过MigR1-CD19-CAR逆转录病毒载体转染人原代T细胞，并通过延长离心转染时间、根据扩增倍数确定个体转染时机而成功优化转染效率，RT-PCR检测CD19-CAR目的基因在转染后人T细胞中高效特异性转录，CD19-CAR-T细胞特异性识别靶细胞引发IFN-γ、TNF-α表达水平增加。