CD38基因敲除的淋巴瘤细胞株构建  

Construction of CD38 Knockout Lymphoma Cell Lines

作　　者：刘秀盈 于梦圆 冯娅茹 宋志茹 刘静静 王建勋 LIU Xiu-ying;YU Meng-yuan;FENG Ya-ru;SONG Zhi-ru;LIU Jing-jing;WANG Jian-xun(School of Life Sciences,Beijing University of Chinese Medicine,Bejing,102488,China;Shenzhen Cell Valley Biopharmaceutical Co.,Ltd,Shenzhen,Guangdong,518118,China;Shenzhen Research Institute,Beijing University of Traditional Chinese Medicine,Shenzhen,Guangdong,518118,China)

机构地区：[1]北京中医药大学生命科学学院,北京102488 [2]深圳细胞谷生物医药有限公司,广东深圳518118 [3]深圳北京中医药大学研究院,广东深圳518118

出　　处：《现代生物医学进展》2024年第10期1807-1812,共6页Progress in Modern Biomedicine

基　　金：高层次人才科研启动经费项目(9011451310032)。

摘　　要：目的:构建Luc^(+)CD38^(-)的Raji细胞株,并进行功能的初步验证,为后期探索淋巴瘤细胞CD38位点免疫逃逸现象奠定基础。方法:通过CRISPR-cas9技术和Piggy Bac(PB)转座子系统,对Luc^(+)Raji细胞的CD38基因位点进行敲除,构建Luc^(+)CD38^(-)Raji细胞株,使用流式细胞术检测与Luc^(+)CD38^(-)Raji细胞株以1:1的比例共孵育CD19 CAR-T和CD38 CAR-T以及未转导的原始T细胞表面活化因子CD69的表达水平,荧光素酶检测法检测上述几组效应细胞对Luc^(+)CD38^(-)Raji细胞株的杀伤效率。结果:成功构建Luc^(+)CD38^(-)Raji细胞,激活实验结果显示,CD19 CAR-T与CD38 CAR-T均可以被Luc^(+)Raji细胞激活。而Luc^(+)CD38^(-)Raji19号单克隆细胞由于缺失CD38的表达,仅能够激活CD19 CAR-T。杀伤实验结果显示,两种CAR-T细胞均能够对Luc^(+)Raji细胞进行杀伤,而CD38 CAR-T对Luc^(+)CD38^(-)Raji19号单克隆细胞的杀伤效率与原始的T细胞相似。结论:成功构建了Luc^(+)CD38^(-)Raji细胞株,为后期探索淋巴瘤CD38位点免疫逃逸现象奠定基础。Objective:To construct a Luc^(+)CD38^(-)Raji cell line and perform the preliminary validation of its function to lay the foundation for the later exploration of the immune escape phenomenon at the CD38 locus in lymphoma cells.Methods:The CD38 gene locus of Luc^(+)Raji cells was knocked down by CRISPR-cas9 technology and Piggy Bac(PB)transposon system,and the Luc^(+)CD38^(-)Raji cell line was constructed,which was assayed by using flow cytometry to co-incubate with the Luc^(+)CD38^(-)Raji cell line at a 1:1 ratio of CD19 CAR-T and CD38 CAR-T as well as the expression level of the surface activation factor CD69 on untransduced primary T cells,and luciferase assay was used to detect the killing efficiency of the above groups of effector cells against the Luc^(+)CD38^(-)Raji cell line.Results:Luc^(+)CD38^(-)Raji cells were successfully constructed,and the results of activation experiments showed that both CD19 CAR-T and CD38 CAR-T could be activated by Luc^(+)Raji cells.While Luc^(+)CD38^(-)Raji19 monoclonal cells were only able to activate CD19 CAR-T due to the lack of CD38 expression.The results of killing experiments showed that both CAR-T cells were able to kill Luc^(+)Raji cells,and the killing efficiency of CD38 CAR-T on Luc^(+)CD38^(-)Raji19 monoclonal cells was similar to that of the original T cell.Conclusions:The Luc^(+)CD38^(-)Raji cell line was successfully constructed,which lays the foundation for exploring the immune escape phenomenon at the CD38 locus of lymphoma at a later stage.

关 键 词：淋巴瘤 CD38 Raji CAR-T CRISPR-cas9 PB转座子 

分 类 号：R-33[医药卫生] R733.4