【摘要】 目的研究双嵌合抗原受体T细胞（BiCAR-T）对人脑胶质瘤细胞系U87的体外细胞毒活性,及其对BALB/c nude裸小鼠恶性胶质瘤模型的体内抗肿瘤作用。方法用BALB/c Nude裸小鼠U87细胞皮下肿瘤模型和颅内肿瘤模型观察Bi CAR-T细胞体内抑瘤作用。皮下肿瘤模型实验分为对照组、模型组和实验组,观察各组小鼠的肿瘤体积生长情况,取肿瘤组织进行病理学检测。颅内肿瘤模型实验分为对照组、模型组和实验组,取肿瘤组织进行病理学检测,用免疫组织化学法检测Cyclin D1的表达情况。结果流式细胞术检测结果表明,BiCAR-T细胞CAR的表达率超过50%。体外实验中,效靶比为5∶1,10∶1,20∶1时:实验组细胞对U87细胞的杀伤率为（21.21±1.11）%,（42.45±2.52）%,（62.34±4.17）%,对照组细胞对U87细胞的杀伤率为（14.15±0.88）%,（22.31±1.21）%,（35.98±2.45）%;实验组IFN-γ水平为（403.36±25.98）,（1756.29±103.36）,（2784.52±190.13）pg·mL^-1,对照组IFN-γ水平为（369.76±22.56）,（1005.56±44.65）,（1954.44±112.04）pg·mL^-1,在效靶比为10∶1和20∶1时,2组间差异有统计学意义（P<0.05）。BiCAR-T细胞对BALB/c nude裸小鼠皮下恶性胶质瘤生长抑制率为45.80%,与模型组的100.00%比较,差异均有统计学意义（P<0.05）。皮下肿瘤模型实验组与模型组相比,可见肿瘤细胞显著减少。颅内肿瘤模型实验组与模型组相比,肿瘤细胞显著减少,Cyclin D1表达下降。结论 BiCAR-T细胞是一种经基因工程修饰的高效靶向的免疫细胞,具有较强的体内外抑制恶性胶质瘤生长的作用。更多还原

【Abstract】 Objective To study the cytotoxicity of two chimeric antigen receptors modified T lymphocytes（ BiCAR-T） on the human glioma cell line U87 in vitro and the anti-tumor effect of Bi CAR-T on the BALB/c nude mouse model of malignant glioma in vivo. Methods Bi CAR-T were prepared and the expression of CARs were analyzed by flow cytometry. In vitro experiment,experiment group（ BiCAR-T cells） and control group（ T lymphocytes） were set up to observe their cytotoxicity on U87 cells by cell count kit（ CCK）-8 assay and interferon-γ（ IFN-γ） secretion when co-culturing with U87 cells for 48 h by enzyme linked immunosorbent assay（ ELISA） at different effect/target ratios（ 5∶ 1,10∶ 1,20∶ 1）. The BALB/c nude mice subcutaneous tumor model was established by the subcutaneous injection of U87 cells and the intracranialtumor model was adopted to assess the anti-tumor effect. In the subcutaneous tumor model study,the mice were randomly divided into control group,model group and experiment group. The tumor volume was calculated and the pathological changes in tumor tissues were observed for comparison. In intracranial tumor model study,the mice were randomly divided into control group,model group and experiment groups. The pathological changes in tumor tissues were observed,and the expression of Cyclin D1 was detected by immunohistochemical staining technique. Results Two CAR expression rates of Bi CAR-T were more than 50%. In vitro experiments proved that the killing rate of effect/target ratios of 5 ∶ 1, 10 ∶ 1 and 20 ∶ 1 in experiment group were（ 21. 21 ± 1. 11） %,（ 42. 45 ± 2. 52） %,（ 62. 34 ± 4. 17） %,and the killing rates of control group were（ 14. 15 ± 0. 88） %,（ 22. 31 ± 1. 21） %,（ 35. 98 ± 2. 45） %,while the IFN-γ secretion of experiment group were（ 403. 36 ± 25. 98）,（ 1756. 29 ± 103. 36）,（ 2784. 52 ± 190. 13） pg · mL^-1, and the IFN-γ secretion of control group were（ 369. 76 ± 22. 56）,（ 1005. 56 ± 44. 65）,（ 1954. 44 ± 112. 04） pg·mL-1. There were significant differences between this two groups at the effect/target ratios of 10∶ 1 and 20∶ 1（ P < 0. 05）. The Results obtained from the in vivo experiments showed that the tumor volumes in experiment group of the subcutaneous tumor model study shrank 45. 80%,compared with the model group 100. 00%（ P < 0. 05）. The pathological changes of tumor tissues showed that the tumor cells in experiment group of the subcutaneous tumor model study and the experiment group of the intracranial tumor model study were significantly decreased. The expression of Cyclin D1 in experiment group of the intracranial tumor model study were significantly decreased. Conclusion The experimental results showed that BiCAR-T can significantly inhibit the growth of glioma provide more evidences to further study the effective targeting therapy on glioma.更多还原