甲基丙烯酰化明胶3D培养的CAR-T细胞靶向杀伤脑胶质瘤细胞的研究  

Study on targeted killing effect of CAR-T cells cultured with methacryloylated gelatin 3D on glioma cells

作　　者：高建 王小倩 杭殿成 柴新 郭威才 朱玉辐 GAO Jian;WANG Xiaoqian;HANG Diancheng(Department of Neurosurgery,the Affiliated Hospital of Xuzhou Medical University,Xuzhou 221006,China)

机构地区：[1]徐州医科大学附属医院神经外科,徐州221006 [2]徐州医科大学神经系统疾病研究所

出　　处：《临床神经外科杂志》2023年第4期401-406,共6页Journal of Clinical Neurosurgery

基　　金：徐州市医学领军人才培养项目(XWRCHT20210031)。

摘　　要：目的研究在甲基丙烯酰化明胶中培养的CAR-T细胞功能及活性的变化,以及用GelMA为载体,负载缓释CAR-T细胞的可行性。方法基因编辑靶向表皮生长因子受体(EGFR)的第二代CAR-T细胞,制备负载CAR-T的甲基丙烯酸酯明胶,通过水凝胶成胶过程图像、储存及损耗模量验证水凝胶成功制备;使用荧光显微镜荧光成像、流式细胞分析验证CAR-T的成功转染;应用CCK-8实验检测水凝胶的生物相容性;构建共培养模型,利用Elisa试剂盒、细胞毒性LDH试剂盒检测上清液中释放的细胞因子、LDH,检测CAR-T是否成功活化,评估CAR-T细胞杀伤效应。结果荧光显微镜下观察转染第4天的CAR-T细胞,见CAR-T细胞聚集成团并散发绿色荧光;第9天流式细胞术示CD3+T细胞的慢病毒转染率为42.2%,CD8+T细胞:CD4+T细胞约为1∶1,CAR-T细胞成功制备。CCK8实验示水凝胶无毒性。Elisa试剂盒示上清液中细胞活性因子IFN-γ释放量显著增多,提示CAR-T细胞识别EGFR抗体后,可成功活化。当效靶比为1∶1时,U87细胞毒性为71.75%,而U87 EGFRvⅢ细胞毒性为18.13%。结论甲基丙烯酸酯化明胶负载培养的CAR-T细胞活性良好,功能正常,可设计使用该水凝胶负载递送CAR-T细胞,实现CAR-T细胞的精确靶向治疗。Objective To study the changes of function and activity of CAR-T cells cultured in methacryloylated gelatin and to explore the feasibility of loading sustained-release CAR-T cells with GelMA as carrier.Methods CAR-T loaded methacrylate gelatin was prepared by gene editing targeting the second generation CAR-T cells of epidermal growth factor receptor(EGFR).The successful preparation of hydrogel was verified by hydrogel formation process image,storage and loss modulus.The successful transfection of CAR-T was verified by fluorescence microscope fluorescence imaging and flow cytometry analysis.CCK-8 test was used to detect the biocompatibility of hydrogel.The co-culture model was constructed,and the cytokines and LDH released from the supernatant were detected by Elisa kit and cytotoxic LDH kit,to detect whether CAR-T was activated successfully and evaluate the killing effect of CAR-T cells.Results The CAR-T cells transfected on the 4th day were observed under the fluorescence microscope,and the CAR-T cells gathered into clusters and emitted green fluorescence.On the 9th day,flow cytometry showed that the lentivirus transfection rate of CD3+T cells was 42.2%.CD8+T cells:CD4+T cells were about 1∶1.CCK8 test showed that the hydrogel was non-toxic.Elisa kit showed a significant increase in the release of cytoactive factor IFN-γin the supernatant,suggesting that CAR-T cells could be activated successfully after recognizing EGFR antibody.With a target ratio of 1∶1,U87 cytotoxicity was 71.75%,while U87 EGFRvIII cytotoxicity was 18.13%.Conclusions The CAR-T cells loaded with methacrylate gelatin have good activity and normal function.The hydrogel can be designed to deliver CAR-T cells to achieve accurate targeted therapy of CAR-T cells.

关 键 词：甲基丙烯酰化明胶 CAR-T 3D细胞培养 胶质瘤 表皮生长因子受体 

分 类 号：R739.41[医药卫生—肿瘤]