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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 被引量:3

Construction of CD19-CAR retroviral vector and modification of its transduction of human T- lymphocytes
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摘要 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.
出处 《中华血液学杂志》 CAS CSCD 北大核心 2015年第4期331-336,共6页 Chinese Journal of Hematology
基金 基金项目:国家自然科学基金(81470322、81172249)
关键词 受体 抗原 质粒 MigR1 转染 T淋巴细胞 细胞因子类 Receptors, antigen Plasmids, MigR1 Transfection T-Lymphocyte Cytokine
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