[CD19嵌合抗原受体逆转录病毒载体的构建及其对人T淋巴细胞转导的修饰]
[Construction of CD19-CAR retroviral vector and modification of its transduction of human T-lymphocytes].
作者信息
Wang Yang, Tang Gusheng, Xu Lili, Ruan Jie, Cheng Hui, Zhou Hong, Hua Yifei, Hu Xiaoxia, Gu Haihui, Qian Baohua, Wang Jianmin, Yang Jianmin
机构信息
Department of Hematology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
出版信息
Zhonghua Xue Ye Xue Za Zhi. 2015 Apr;36(4):331-6. doi: 10.3760/cma.j.issn.0253-2727.2015.04.016.
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
通过逆转录病毒载体提高包含靶向CD19的单链可变区（scFv）的MigR1-CD19嵌合抗原受体（CAR）对T淋巴细胞的转导效率。
方法
通过重组DNA技术将CD19-CAR片段插入逆转录病毒载体（MigR1），转染plat-A包装细胞系后，收集病毒上清转导K562细胞系和活化的人T淋巴细胞。我们用流式细胞术测定转导效率，用RT-PCR确认CD19-CAR基因的转录。在酶联免疫吸附（ELISA）试验中检测转导的T细胞以CD19特异性方式产生IFN-γ和TNF-α的能力。
结果
（1）使用MigR1-CD19-CAR逆转录病毒载体产生高滴度逆转录病毒。（2）MigR1-CD19-CAR对K562细胞系的转导效率显著高于人T淋巴细胞（P<0.01）。（3）120分钟离心可显著提高T淋巴细胞的转导效率至（54.5±14.6）%。（4）根据T淋巴细胞体外增殖倍数个体化确定转导时间可提高转导效率，本研究中最高转导效率为69.3%。CD19-CAR基因序列高效特异性转录。（5）与CD19-K562细胞共培养时，CD19-CAR转导的T淋巴细胞释放的IFN-γ和TNF-α显著增加至（13 230±1 543）pg/ml和（4 217±211）pg/ml。
结论
我们成功构建了一种通过名为MigR1的逆转录病毒载体靶向CD19的第二代CAR（MigR1-CD19-CAR）。根据T淋巴细胞体外增殖倍数个体化确定转导时间和120分钟离心可提高T淋巴细胞的CAR转导效率。RT-PCR证实CD19-CAR基因高效特异性转录。CD19-CAR转导的T淋巴细胞在被靶细胞激活时释放的IFN-γ和TNF-α显著增加。
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