双嵌合抗原受体T细胞靶向治疗急性髓系白血病的实验研究

Experimental study on bi-chimeric antigen receptors modified T lymphocytes targeting on acute myeloid leukemia

目的 研究双嵌合抗原受体T细胞（BiCAR-T细胞）对人急性髓系白血病细胞株HL60的体外细胞毒活性,以及其对NOD SCID小鼠急性髓系白血病模型的体内抗肿瘤作用.方法 制备BiCAR-T细胞,用流式细胞仪检测嵌合抗原受体（CAR）的表达情况.体外实验分为实验组（BiCAR-T细胞）和对照组（T淋巴细胞）,设立不同的效靶比（5∶1、10∶1、20∶1）,用CCK8法测定BiCAR-T细胞体外对HL60细胞的杀伤率,用酶联免疫吸附试验（ELISA）法检测BiCAR-T细胞与HL60细胞共培养48 h后培养上清液中干扰素-γ（IFN-γ）水平.用HL60细胞尾静脉注射NOD SCID小鼠建立急性髓系白血病模型观察BiCAR-T细胞体内抑瘤作用,按随机数字表法将NOD SCID小鼠随机分为空白对照组、模型组和治疗组,每组10只.空白对照组通过尾静脉注射0.9％ NaCl,模型组和治疗组通过尾静脉注射1×107个HL60细胞造模,造模20 d后治疗组经尾静脉注射2×107个BiCAR-T细胞,空白对照组和模型组经尾静脉注射0.9％ NaCl,每周注射3次,共2周,注射体积均为0.2 ml.治疗2周后取小鼠脏器进行病理学检测.结果 流式细胞术检测结果表明BiCAR-T细胞CAR的表达率＞50.00％.效靶比为5∶1、10∶1、20∶1时,实验组BiCAR-T细胞和对照组T淋巴细胞的杀伤率分别为（25.43±1.32）％∶（16.18±0.75）％、（50.33±3.11）％∶（25.47±1.27）％和（85.89±3.96）％∶（49.45±2.77）％,其中BiCAR-T细胞和T淋巴细胞对HL60细胞杀伤率的主效应比较,差异有统计学意义（F=404.17,P＜0.001）;不同效靶比时BiCAR-T细胞和T淋巴细胞对HL60细胞杀伤率比较,差异有统计学意义（F=548.09,P＜0.001）;不同效靶比时BiCAR-T细胞对HL60细胞的杀伤效率均显著优于T淋巴细胞（F=45.36,P＜0.001）.实验组BiCAR-T细胞和对照组T淋巴细胞与HL60细胞共培养48 h后培养上清液中IFN-γ水平分别为（435.65 ±20.44） pg/ml∶（356.75±19.87） pg/ml、（1 639.98±95.75） pg/ml∶（1 109.37±80.98） pg/ml和（3 467.43±187.54） pg/ml∶（2 245.52±112.66） pg/ml,其中实验组和对照组IFN-γ水平的主效应比较,差异有统计学意义（F=156.24,P＜0.001）;不同效靶比时IFN-γ水平比较,差异有统计学意义（F=857.67,P＜0.001）;不同效靶比时实验组的IFN-γ水平均显著高于对照组（F =46.31,P＜0.001）.对小鼠肝、脾组织进行苏木精-伊红（HE）染色,结果显示治疗组的白血病细胞浸润程度显著低于模型组.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,能于体内外强效抑制急性髓系白血病细胞的浸润.

 Objective To study the cytotoxicity of bi-chimeric antigen receptors modified T lymphocytes （BiCAR-T） on the human acute myeloid leukemia （AML） cell line HL60 in vitro and the anti-tumor effects of BiCAR-T on the NOD SCID mouse model of AML in vivo.Methods The BiCAR-T were prepared and the expression of chimeric antigen receptor （CAR） of prepared BiCAR-T was analyzed by flow cytometry.In vitro study was divided into two groups：the experiment group （BiCAR-T） and the control group （T lymphocyte）.The killing rate of BiCAR-T in vitro on HL60 cells was determined by CCK8 assay and the level of interferon-γ （IFN-γ） secreted from BiCAR-T co-culturing with HL60 cells for 48 hours was detected by enzyme linked immunosorbent assay （ELISA） at different effect/target ratios （5∶1,10 ∶ 1,20 ∶ 1）.The NOD SCID mice AML model was established by the injection of HL60 cells through tail vein and used to assess the antitumor effects in vivo.The mice were randomly divided into three groups according to the random number table：the blank control group receiving 0.9% NaCl 0.2 ml through tail vein,the model group and the treatment group receiving 1 × 107 HL60 cells in 0.2 ml phosphate buffer saline （PBS）.After 20 days,the treatment group was injected with 2 × 107BiCAR-T in 0.2 ml PBS 3 times a week for 2 weeks,while the other two groups received 0.9% NaCl 0.2 ml.The pathological changes in the mice livers and spleens were observed after 2 weeks of treatment.Results The CAR expression rates of BiCAR-T were more than 50.00%.In vitro experiments proved that the killing rates of BiCAR-T in the experimental group and T lymphocytes in the control group on HL60 cells were （25.43 ±1.32）% vs.（16.18 ±0.75）%,（50.33±3.11）% vs.（25.47±1.27）%,and （85.89 ± 3.96） % vs.（49.45 ± 2.77） % at different effect/target ratios （5 ∶ 1,10 ∶ 1,20 ∶ 1）.The killing efficiency of BiCAR-T and T lymphocytes on HL60 cells was significantly different （F =404.17,P 〈 0.001）;the killing efficiency of BiCAR-T and T lymphocytes on HL60 cells was significantly different at different effect/ target ratios （F =548.09,P 〈 0.001）;and the killing efficiency on HL60 cells in the experimental group （BiCAR-T） was significantly higher than that in the control group （T lymphocytes） at different effect/target ratios （F =45.36,P 〈 0.001）.The IFN-γlevels secreted from BiCAR-T in the experiment group and T lymphocytes in the control group co-culturing with HL60 ceils after 48 h were （435.65 ± 20.44） pg/ml vs.（356.75 ± 19.87） pg/ml,（1 639.98 ± 95.75） pg/ml vs.（1 109.37 ± 80.98） pg/ml,and （3 467.43 ± 187.54）pg/ml vs.（2 245.52 ± 112.66）pg/ml.The IFN-γlevel in the experiment group （BiCAR-T） and the control group （T lymphocytes） was significantly different （F =156.24,P 〈 0.001）;the IFN-γ level was significantly different at different effect/target ratios （F =857.67,P 〈 0.001）;the IFN-γlevel in the experimental group （BiCAR-T） was significantly higher than that in the control group （T lymphocytes） at different effect/ target ratios of 5 ∶ 1,10 ∶ 1,20 ∶ 1,respectively （F =46.31,P 〈 0.001）.The result of hematoxylineosin staining （HE） staining showed that leukocyte infiltration in the treatment group was significantly decreased compared with the model group.Conclusion The experimental results showed that BiCAR-T is a kind of efficient targeted immunocyte modified by gene engineering,and it can significantly inhibit leukocyte infiltration of AML in vivo and in vitro.