摘要
CAR-T细胞疗法通过靶向识别肿瘤细胞表面抗原,特异性杀伤肿瘤细胞,近年来已经成为肿瘤免疫疗法的研究热点。通过基因工程方法构建靶向人类表皮生长因子受体2(HER2)的CAR慢病毒表达质粒,以磷酸钙沉淀辅助多质粒共转染HEK293T细胞包装,制备CAR慢病毒颗粒lenti-car,感染人外周血单核细胞获得HER2靶向的CAR-T细胞,并分析其对HER2阳性和阴性肿瘤细胞的特异性抑制效果。研究结果表明,构建的CAR-T细胞可被HER-2阳性的肿瘤细胞特异性激活,分泌大量炎症性细胞因子IFN-γ和IL-2。在同样效靶比等处理条件下,构建的HER2靶向CAR-T细胞对HER2阳性的人卵巢癌细胞株SK-OV-3的生长抑制率为(58.47±1.72)%,显著高于对照组(P<0.05);而对HER2阴性的人慢性髓原白血病细胞株K562的生长抑制率为(11.74±2.37)%,与对照组无显著差异(P>0.05)。进一步,在K562细胞中转染人HER2表达载体使其成为HER2阳性,则HER2靶向CAR-T细胞对其的生长抑制率上升为(30.41±7.59)%,较HER2阴性K562具有明显差异(P<0.05)。研究结果表明,构建的HER2靶向的第二代CAR-T细胞可选择性地抑制高表达HER2蛋白的肿瘤细胞的生长,暗示了其对HER2阳性肿瘤进行细胞免疫治疗的临床应用前景。
CAR-T cell therapy that targets surface antigens to kill tumor cells specifically has recently become another cornerstone in tumor immunotherapy. In this study, a lentiviral expression plasmid of CAR targeting human epidermal growth factor receptor 2（HER2） was constructed by genetic engineering. The recombinant plasmid was co-transfected with other packaging plasmids into HEK293 T cells by calcium phosphate precipitation to generate lenti-car, which are CAR lentiviral particles. HER2-specific CAR-T cells were obtained by transducing human peripheral blood mononuclear cells with lenti-car. Their specific inhibitory effects on HER2-positive and HER2-negative tumor cells were analyzed in vitro. The constructed CAR-T cells were specifically activated by HER2-expressing tumor cells as indicated by secretion of IFN-γ and IL-2. The inhibitory rate on HER2-positive SK-OV-3 cell line was（58.47±1.72）%, significantly higher than that on the mock-treated control group（P〈0.05）. The inhibitory rate on HER2-negative K562 cell lines was（11.74±2.37）%, which was not significantly different from that on the control group（P〉0.05）. Furthermore, when we transfected a HER2-expressing vector into K562, the inhibitory rate increased to（30.41±7.59）%, which was higher than that on HER2-negative K562（P〈0.05）. Thus, the constructed second-generation HER2-specific CAR-T cells specifically suppressed growth of tumor cells overexpressing HER2 protein, suggesting that HER2-specific CAR-T cells might prove useful for immunotherapy of HER2-positive cancer.
作者
李永强
姚崧源
李延胜
徐明恺
张惠文
张成刚
Yongqiang Li1,2, Songyuan Yao1,2, Yansheng Li1,2, Mingkai Xu1, Huiwen Zhang1, and Chenggang Zhang1(1 Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, Liaoning, China; 2 University of Chinese Academy of Sciences, Beijing 100049, Chin)
出处
《生物工程学报》
CAS
CSCD
北大核心
2018年第5期731-742,共12页
Chinese Journal of Biotechnology
基金
中国科学院战略性先导科技专项(A类)(No.XDA12020225)
沈阳市科技计划项目(Nos.Z17-7-013
F14-150-9-00)
国家科技重大专项重大新药创制项目(No.2012ZX09102301-013)资助~~
