利用MigR1质粒构建CD19-CAR逆转录病毒载体优化其对人原代T细胞的转染效率

Construction of CD19-CAR retroviral vector and modification of its transduction of human T- lymphocytes

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构建人逆转录病毒载体，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120min组的转染率为（54．5±14．6）％，明显高于未离心组、离心30min组及离心60min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72hT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（13230±1543）pg／ml、（4217±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。

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 CD 19-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）Transductionefficiency 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 rain 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-lymphoeytes significantly increased when activated by target cells.