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-Tcell]，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pCDH-GPC3-CAR，感染T细胞；采用Westernblotting、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pCDH-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pCDH-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)%,P<0.01]。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，P<0.01]，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。

Objective: To construct chimeric antigen receptor (CAR)-modified T cell (CAR-T) which targets GPC3 positive hepatoma cell, and to assess its killing efficacy to the GPC3 positive hepatoma cell. Methods: To enhance expression efficiency of the CAR molecule, technique for favored codon and modification of gene sequence were se-ected. CAR gene targeting GPC3 antigen was designed and lentiviral vector carrying GPC3-CAR gene, pCDH-GPC3-CAR, was constructed, which was transfected into T cell. Western blotting, flow cytometry, real-time cellanal-ysis (RTCA) and ELISA assays were used respectively to detect expression of GPC3-CAR molecule in the CAR-T cell, infection efficiency of the lentivirus to T cells, killing activity and specificity of the GPC3-CAR-T cell to the GPC3 positive hepatoma cell. Results: The gene segment in the pCDH-GPC3-CAR recombinant lentivirus plasmid,molecular mass of which is the same as the GPC3-CAR molecule, was found in a result of double enzyme digestion,indicating that the pCDH-GPC3-CAR lentivirus plasmid was successfully constructed. About 54.38% of the GPC3-T cell expressing GPC3-CAR molecule were celled as the GPC3-CAR-T cell. Killing efficacy of the GPC3-CAR-T cell to the GPC3 positive Huh-7 hepatoma cell was higher than that to the GPC3 negative SK-HEP-1 cell ([78.96±4.76]% vs [6.87±3.15]%). IFN-γ secretion efficiency of the GPC3-CAR-T cell co-cultured with the GPC3 positive Huh-7 hepatoma cell in the CAR-T group was higher than that in the Mock group ([21 371.4±1 808.3]pg/ml vs [152.8±12.5] pg/ml), which could be one of the mechanisms of high efficacy in killing hepatoma cells. Conclusion:The GPC3-CAR-T cell targeting hepatoma cells might have high efficacy ability to secrete cytokine IFN-γ and spe-cific, higher efficient ability to kill the GPC3 positive hepatoma cell, which would lay a foundation to carry further forward pre-clinical and clinical research on the GPC3-CAR-T cell.

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）