【摘要】 目的 制备特异性杀伤人类表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)表达阳性的膀胱癌细胞的嵌合抗原受体T细胞（chimeric antigen receptor T-Cell,CAR T）,初步探索HER2-CAR T细胞治疗膀胱癌的可行性。方法 首先免疫组化法检测膀胱癌患者石蜡组织切片中肿瘤细胞HER2表达情况，然后以赫赛汀单克隆抗体的单链抗体（single chain antibody fragment,scFv）序列作为CAR T的识别序列，构建包含铰链，共刺激分子（CD28和4-1BB胞内域）和CD3z的三代CAR T慢病毒质粒。使用293T细胞包装慢病毒，感染人源外周血单个核细胞（peripheral blood mononuclear cell, PBMC）细胞，制备HER2-CAR T细胞，并采用流式细胞术检测CAR的转导效率。将CAR T细胞分别与HER2表达阳性并且稳转构建表达荧光素酶的SK-BR3阳性对照细胞和MDAMB-468阴性对照细胞及T24膀胱癌细胞共培养，采用荧光素酶报告基因检测法检测杀伤效果，利用酶联免疫吸附测定（enzyme linked immunosorbent assay,ELISA）测定CAR T细胞与癌细胞共孵育时干扰素(Interferon gamma, IFN-)的分泌水平。结果 免疫组化法检测显示多数膀胱癌患者的病理组织高表达HER2。免疫荧光和流式细胞荧光分选技术(fluorescence-activated cell sorting,FACS)显示T24膀胱癌细胞高表达HER2。流式细胞术检测显示T细胞的CAR转导效率为69%，体外共培养实验显示CAR T细胞与T24细胞比为5:1时，杀伤效率达到73%。CAR T细胞对SK-BR3细胞和T24细胞具有较强毒性，但是对于MDA-MB-468阴性对照细胞仅在高效靶比时有较弱杀伤，显示了HER2-CAR T细胞杀伤的特异性。共孵育时，SK-BR3组培养基上清中INF-浓度达到319 pg/mL, T24组培养基上清中INF-浓度为275 pg/mL。结论 成功制备靶向杀伤HER2阳性膀胱癌细胞的CAR T细胞，为CAR T细胞治疗膀胱癌提供了初步的研究支持。更多还原

【Abstract】 Objective To establish a method of CAR T therapy for HER2 expressing bladder cancer, and explore the potential of the CAR T therapy for this disease. Methods HER2 expression was examined using specimen from patients with bladder cancer by immunohistochemistry(IHC). The CAR was constructed into a third generation of lentivirus backbone, which consists of a single-chain variable fragment(scFV) derived from Herceptin monoclonal antibody, a hinge, a dual costimulatory domain(4-1BB and CD28), and a CD3z intracellular domain. The 293T cells were used for lentivirus production. The PBMC(Peripheral blood mononuclear cell) was transduced with this virus to produce HER2-CAR T cells, followed by flow cytometry for detection of transduction efficiency. Co-culture of CAR T cells with SKBR3(positive control), MDA-MB-468(negative control) or bladder cancer cell line T24 was performed to evaluate the cytotoxic effect via luciferase activity assay. The INF-release was measured by ELISA. Results The IHC result showed that HER2 was highly expressed in most of the specimens. The immune fluorescence staining and FACS showed that T24 cell line expressed high level of HER2. The transduction efficiency of CAR for T cells was 69%. The coculture experiment demonstrated 73% of specific lysis when the ratio of CAR T: T24 was 5:1. Although CAR T cells displayed cytotoxic activity to SK-BR3 and T24 cell lines, it had only a minimal killing effect on the HER2 negative cell line MDAMB-468 at high E:T ratio, indicating high specificity of the HER2-CAR T cells. The concentrations of INF-in the supernatant were 319 pg/mL(SK-BR3) and 275 pg/mL(T24) as measured by ELISA. Conclusion We have established a CAR T therapy method for specific killing of HER2 positive bladder cancer and have provided preliminary evidence for application of this technology for bladder cancer therapy.更多还原