基于蛋白L染色方法的流式细胞术检测嵌合抗原受体的表达  

A novel staining protocol based on protein L for detection of expression of chimeric antigen receptor by flow cytometry

作　　者：邓海峰[1] 韩为东[2] 蒋敬庭[1] DENG Haifeng;HAN Weidong;JIANG Jingting(Department of Tumor Biological Treatment, the Third Affiliated Hospital, Soochow University, Changzhou 213003, Jiangsu;Department of Molecular Biology and Immunology, Chinese PLA General Hospital, Beijing 100853, China)

机构地区：[1]苏州大学附属第三医院肿瘤生物诊疗中心江苏省肿瘤免疫治疗工程技术研究中心苏州大学细胞治疗研究院,江苏常州213003 [2]解放军总医院分子免疫学研究室,北京100853

出　　处：《临床检验杂志》2018年第4期241-244,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家科技支撑计划(2015BAI12B12);国家自然科学基金(31570877;31570908);海外及港澳学者合作研究基金(3172001);江苏省肿瘤免疫治疗工程技术研究中心(BM2014404)

摘　　要：目的探讨以蛋白L(protein L)为基础的流式细胞染色方法用于检测患者T细胞表面嵌合抗原受体(chimeric antigen receptor,CAR)表达的技术可行性。方法单采2例CD19阳性淋巴瘤患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),磁珠分选法获得T细胞,加入靶向CD19、CD22的CAR逆转录病毒,转染、诱导培养成CAR-T细胞。通过生物素化蛋白L(biotinylated protein L)-亲合素(streptavidin-PE)系统对CAR-T细胞表面的单链抗体片段(single-chain variable fragment,sc Fv)进行标记,用流式细胞术检测活化培养的T细胞中CD19-CAR、CD22-CAR表达阳性T细胞的比例,以常规Anti-Ig G染色的流式细胞检测方法作为对照组,进行结果对比。结果 sc Fv-biotinylated protein L-streptavidin-PE-流式细胞染色方法能够检测到CAR-T细胞表面CAR表达。2例患者蛋白L实验组和Anti-Ig G对照组CD19-CAR-T细胞比例分别为71.6%vs64.2%和49.3%vs 43.8%;CD22-CAR-T细胞比例分别为53.1%vs 46.3%和56.5%vs 64.0%,测定结果相似。结论以蛋白L为基础的染色方法可以作为流式细胞术检测CAR-T细胞的常规染色方法。Objective To evaluate the feasibility of the novel staining protocol based on protein L in flow cytometry to detect the expression of chimeric antigen receptor （CAR） on the surface of T cells from patients. Methods The peripheral blood mononuclear cells（PBMCs） were collected from 2 patients with CD19 ＋ lymphoma by hemapheresis and T cells were purified by magnetic bead selection. CAR was transfected with retroviruses targeting CD19 and CD22 into T cells to induce chimeric antigen receptor-modified T cells (CAR-T cells）. The single-chain fragments of antibody molecules on the surface of CAR-T ceils were labeled by biotinylated pro-tein L-streptavidin-PE system. The proportions of activated CD19-CAR and CD22-CAR positive T cells in the culture were detected by flow cytometry. The results were compared with those detected by conventional flow cytometry method based on Anti-IgG staining. Results The expression of CAR on the surface of CAR-T cells was successfully detected by flow cytometry protocol based on the staining of single-chain variable fragment （scFv）-biotinylated protein L-streptavidin-PE. The percentages of CD19-CAR-T cells from the 2 patients were 71.6% vs 64.2% and 49.3% vs 43.8% in the protein L group and the Anti-IgG control group respectively, and the percentages of CD22-CAR-T cells were 53.1% vs 46.3% and 56.5% vs 64.0%. The results of the both groups were similar. Conclusion The staining protocol based on protein L could be used as a routine staining method in flow cytometry for the detection of CAR-T cells.

关 键 词：嵌合抗原受体T细胞 蛋白L 流式细胞术 

分 类 号：R446.6[医药卫生—诊断学]