靶向肝癌的GPC3-CAR-T细胞的构建与功能鉴定

发布时间：2018-05-13 06:19

本文选题：磷脂酰肌醇蛋白聚糖- + 嵌合抗原受体T细胞　；参考：《中国肿瘤生物治疗杂志》2017年07期

【摘要】：目的:构建靶向GPC3阳性肝癌细胞的嵌合抗原受体修饰T细胞[chimeric antigen receptor-modified T cell,CAR-T cell],并评估其对GPC3阳性肝癌细胞的杀伤效能。方法:选择偏爱密码子技术改造基因序列,以增强CAR分子表达效率,设计靶向GPC3抗原的CAR分子基因并构建携带GPC3-CAR基因的慢病毒表达载体p CDH-GPC3-CAR,感染T细胞;采用Western blotting、流式细胞术、实时细胞监测和ELISA技术分别检测GPC3-CAR分子在CAR-T细胞的表达、慢病毒感染T细胞效率、GPC3-CAR-T细胞对GPC3阳性肝癌细胞的杀伤活性和特异性。结果:双酶切p CDH-GPC3-CAR重组慢病毒质粒可见分子质量和GPC3-CAR分子一致的基因片段,成功构建p CDH-GPC3-CAR慢病毒质粒。约54.38%的GPC3-T细胞表达GPC3-CAR分子,称为GPC3-CAR-T细胞。GPC3-CAR-T细胞对GPC3阳性的肝癌细胞Huh-7比GPC3阴性的肝癌细胞SK-HEP-1具有更高效的杀伤能力[(78.96±4.76)%vs(6.87±3.15)%,P0.01]。与GPC3阳性Huh-7肝癌细胞共培养的肝癌GPC3-CAR-T细胞分泌IFN-γ水平比Mock组高效[(21 371.4±1 808.3)vs(152.8±12.5)pg/ml,P0.01],这可能是其高效杀伤肝癌细胞的机制之一。结论:靶向肝癌的GPC3-CAR-T细胞具有高效分泌IFN-γ细胞因子并特异高效杀伤GPC3阳性肝癌细胞的能力,为进一步推进GPC3-CAR-T临床前和临床研究奠定基础。
[Abstract]:Aim: to construct chimeric antigen receptor modified T cells (chimeric antigen receptor-modified T cell CAR-T cell) targeting GPC3 positive hepatoma cells and evaluate their cytotoxicity to GPC3 positive hepatoma cells. Methods: to select the codon preference technique to modify the gene sequence, to enhance the expression efficiency of CAR, to design the CAR molecular gene targeting the GPC3 antigen and to construct the lentivirus expression vector pCDH-GPC3-CARcarrying the GPC3-CAR gene to infect T cells, and to infect T cells by Western blotting and flow cytometry. The expression of GPC3-CAR molecules in CAR-T cells was detected by real-time cell monitoring and ELISA technique. The cytotoxicity and specificity of GPC3-CAR-T cells infected by lentivirus to GPC3 positive hepatoma cells were investigated. Results: the recombinant lentivirus plasmid of p CDH-GPC3-CAR was digested with double enzyme, and the molecular weight of the recombinant lentivirus plasmid was the same as that of GPC3-CAR molecule. The plasmid of p CDH-GPC3-CAR lentivirus was successfully constructed. About 54.38% of the GPC3-T cells expressed GPC3-CAR molecule, which was called GPC3-CAR-T cell. GPC3-CAR-T cell had a more effective killing effect on GPC3 positive hepatoma cell Huh-7 than GPC3 negative HCC cell line SK-HEP-1 [78.96 卤4.76)%vs(6.87 卤3.15P 0.01]. The level of IFN- 纬 secreted by GPC3-CAR-T cells co-cultured with GPC3 positive Huh-7 hepatoma cells was higher than that of Mock group [21 371.4 卤1 808.3)vs(152.8 卤12.5 808.3)vs(152.8 卤12.5 mg / ml P0.01], which may be one of the mechanisms of its high killing effect on hepatoma cells. Conclusion: GPC3-CAR-T cells targeting HCC have the ability of secreting IFN- 纬 cytokines and killing GPC3 positive hepatoma cells specifically and efficiently, which will lay a foundation for further clinical and clinical study of GPC3-CAR-T.
【作者单位】：第三军医大学大坪医院野战外科研究所创伤、烧伤与复合伤国家重点实验室第一研究室;
【基金】：国家自然科学基金资助项目(No.81502434) 第三军医大学校管课题资助项目(No.2014YQN06)~~
【分类号】：R735.7

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