摘要
目的构建干扰胸腺细胞选择相关高迁移率群体盒(TOX)基因联合抗CD38 CAR-T细胞,观察其对血液肿瘤细胞增殖和凋亡能力的影响。方法采集健康志愿者外周血,分离单个核细胞(PBMC),提取原代T淋巴细胞。构建以shRNA技术靶向抑制TOX的抗CD38 CAR分子,分别命名为TOX-shRNA1-CD38 CAR和TOX-shRNA2-CD38 CAR,另设计一条不针对任何靶点的无意义RNA序列作为对照,命名为CD38 CAR分子。将分子酶切并连接逆病毒载体包装质粒pMFG,获得pMFG-TOX-shRNA1-MYC-CD38 CAR、pMFG-TOX-shRNA2-MYC-CD38 CAR和pMFG-MYC-CD38 CAR。将人原代T淋巴细胞分为CD38 TOX-shRNA1-CD38 CAR-T组、TOX-shRNA2-CD38 CAR-T组、CAR-T组,分别转导pMFG-TOX-shRNA1-MYC-CD38 CAR、pMFG-TOX-shRNA2-MYC-CD38 CAR和pMFG-MYCCD38 CAR质粒,获得CD38 CAR-T细胞、TOX-shRNA1-CD38 CAR-T细胞和TOX-shRNA2-CD38 CAR-T细胞。采用RT-QPCR法检测三组细胞中TOX mRNA的相对表达量;记录三组细胞0~10 d体外培养生长倍数,观察细胞增殖能力。选取CD38高表达的人多发性骨髓瘤荧光素酶标记细胞RPMI-Luc、人Burkitt’s淋巴瘤荧光素酶标记细胞Raji-Luc作为靶细胞,与三组CAR-T细胞分别共培养48 h;另取三组CAR-T细胞单独培养作为对照,不加肿瘤细胞。采用流式细胞术检测三组CAR-T细胞表面CD69的表达效率,评价CAR-T细胞活化情况;对三组细胞进行CFSE染色,采用流式细胞术观察CAR-T细胞增殖能力;荧光素酶化学发光法观察CAR-T细胞在不同效靶比(1∶2、1∶4、1∶8)时对肿瘤细胞的杀伤效率;ELISA法检测三组CAR-T细胞上清中IFN-γ释放量;流式细胞术检测三组细胞表面PD-1表达量,评估CAR-T细胞的耗竭程度。结果 CD38 CAR-T组、TOX-shRNA1-CD38 CAR-T组、TOXshRNA2-CD38 CAR-T组的转导效率分别为41.51%、41.28%、44.84%,均超过40%。与CD38 CAR-T组比较,TOXshRNA2-CD38 CAR-T组TOX mRNA表达水平降低(P <0.05),TOX-shRNA1-CD38 CAR-T组无明显变化(P>0.05)。三组CAR-T细胞体外培养0~10 d均稳定增殖,三组间生长倍数比较差异无统计学意义(P <0.05)。三组与肿瘤细胞共培养后,CD69表达效率均升高(P均<0.01)。与单独培养的CAR-T细胞相比,三组CAR-T细胞与Raji-Luc、RPMI-Luc细胞共培养后FITC信号左移,增殖加快,三组增殖能力比较无统计学差异(P> 0.05)。与CD38 CAR-T组相比,TOX-shRNA2-CD38 CAR-T组在不同效靶比时对肿瘤细胞的杀伤效率均提高,γ干扰素释放水平升高,细胞表面PD-1表达水平降低(P <0.05或<0.01),而TOX-shRNA1-CD38 CAR-T组与CD38 CAR-T组比较,肿瘤细胞的杀伤效率、γ干扰素释放水平、细胞表面PD-1表达水平均无统计学差异(P均>0.05)。结论成功构建了靶向抑制TOX基因联合抗CD38 CAR-T细胞,TOX-shRNA-CD38 CAR-T细胞能够有效抑制TOX表达;经肿瘤细胞激活后,其体外增殖活性及抗肿瘤能力显著增强,且TOX-shRNA-CD38 CAR-T细胞耗竭也得到改善。
Objective To construct the CAR-T cells with inhibition of TOX gene and anti-CD38,and to observe their effects on the proliferation and apoptosis of hematological tumor cells.Methods The peripheral blood of healthy volunteers was acquired,the mononuclear cells(PBMC) were separated,and the primary T lymphocytes were extracted.The antiCD38 CAR molecules targeting TOX inhibition by shRNA technology were constructed and named as TOX-shRNA1-CD38 CAR and TOX-shRNA2-CD38 CAR,respectively,and an unmeaning RNA sequence which was not against any target site was designed to be the control and named as CD38 CAR molecule.The molecule was subject to digestion and connection with reverse virus vector packaging plasmid(pMFG) to obtain pMFG-TOX-shRNA1-MYC-CD38 CAR,pMFG-TOX-shRNA2-MYC-CD38 CAR,and pMFG-MYC-CD38 CAR.The human primary T lymphocytes were divided into the CD38 TOXshRNA1-CD38 CAR-T group,TOX-shRNA2-CD38 CAR-T group,and CAR-T group,and pMFG-TOX-shRNA1-MYCCD38 CAR,pMFG-TOX-shRNA2-MYC-CD38 CAR and pMFG-MYC-CD38 CAR were transduced,separately,to obtain the CD38 CAR-T cells,TOX-shRNA1-CD38 CAR-T cells and TOX-shRNA2-CD38 CAR-T cells.The RT-QPCR was used to detect the relative expression of TOX mRNA in cells of the three groups;the growth multiple of cells cultured in vitro for0-10 days in the three groups was recorded and the cell proliferation was observed.The Raji-Luc and RPMI-Luc cells were used as the target cells to be cultured for 48 h together with CAR-T cells of three groups;CAR-T cells of these three groups were cultured separately to be used as the control without tumor target cells.Flow cytometry was used to detect the expression of CD69 on the surface of three groups to evaluate the activation of CAR-T cells.The CFSE staining method was used to observe the proliferation ability of CAR-T cells.The luciferase chemiluminescence method was used to observe the killing efficiency of CAR T cells against tumor target cells at different effector-target ratios(1∶ 2,1∶ 4,and 1∶ 8).ELISA was used to detect the IFN-γ releasing amount in cell supernatants of these three groups.Flow cytometry was used to detect the PD-1 expression levels of three groups and to assess the depletion degree of CAR-T cells.Results The transduction efficiency of the CD38 CAR-T group,TOX-shRNA1-CD38 CAR-T group,TOX-shRNA2-CD38 CAR-T group,and control group reached to 41.51%,41.28%,and 44.84%,respectively,exceeding 40%.Compared with the CD38 CAR-T group,the TOX mRNA expression level of TOX-shRNA2-CD38 CAR-T group was lowered(P < 0.05) and no obvious change occurred in the TOX-shRNA1-CD38 CAR-T group(P > 0.05).Three groups of CAR-T cells were of stable proliferation after being cultured in vitro for 0-10 d,and there was no statistically significant difference in the growth multiple among the three groups(P < 0.05).After the cells of three groups were cultured together with the tumor target cells,the CD69 expression efficiency increased(P < 0.01).Compared with the CAR-T separate culture group,the FITC signal shifted left and the proliferation was accelerated after CAR-T cells of three groups were cultured together with Raji-Luc and RPMI-Luc cells,and there was no statistical difference in proliferation ability among three groups(P > 0.05).Compared with the CD38 CAR-T group,the killing efficiency of the TOX-shRNA2-CD38 CAR-T group against tumor target cells increased at different effector-target ratios,its γ-interferon release level increased and its PD-1 expression level on the cell surface decreased(P <0.05 or P < 0.01).There were no statistical differences in the killing efficiency against tumor target cells,γ-interferon release level,or PD-1 expression level on the cell surface between the CD38 CAR-T group and the TOX-shRNA1-CD38 CART group(all P > 0.05).Conclusion The CAR-T cells with inhibition of TOX gene and anti-CD38 are successfully constructed,the TOX-shRNA-CD38 CAR-T cells can effectively inhibit the TOX expression,and theirin vitro proliferation activity and anti-tumor activity are remarkably strengthened after the activation by tumor target cells,and the depletion of TOX-shRNA-CD38 CAR-T cells is improved.
作者
宋志茹
刘秀盈
朱晶晶
刘静静
冯娅茹
王建勋
SONG Zhiru;LIU Xiuying;ZHU Jingjing;LIU Jingjing;FENG Yaru;WANG Jianxun(School of Life Sciences,Beijing University of Chinese Medicine,Beijing 102488,China;不详)
出处
《山东医药》
CAS
2022年第2期1-6,17,共7页
Shandong Medical Journal
基金
北京中医药大学高层次人才科研启动经费项目(9011451310032)。
作者简介
第一作者:宋志茹(1993-),女,硕士,主要研究方向为shRNA优化CAR-T疗法。E-mail:S18811360572@163.com;通信作者:王建勋(1973-),男,教授,博士生导师,主要研究方向为现代化基因与细胞治疗手段治疗血液疾病的临床转化。E-mail:jianxun.wang@bucm.edu.cn。
