嵌合抗原受体 (CAR) 修饰的 T 细胞疗法已成为恶性肿瘤治疗领域内的研究重点，催乳素受体 (PRLR) 也被证明其过表达会影响乳腺癌的发生与发展，是乳腺癌治疗的潜在靶点。本研究建立了三质粒系统 [pHIV- 绿色荧光蛋白 (GFP)、psPAX2、pVSVG] 包装慢病毒，并将其应用于新构建的靶向 PRLR 的 CAR 慢病毒表达载体，通过进一步优化慢病毒制备条件，测定慢病毒滴度，成功构建了靶向 PRLR 的 CAR-T 细胞。结果表明，制备慢病毒的最优条件为 ：PRLRCAR ∶ psPAX2 ∶ pVSVG 质量比为 3 ∶ 2 ∶ 1，且和转染试剂以质量比 1 ∶ 2 混合，经浓缩后滴度可达到 1.88×107 TU/ml。以此慢病毒感染人 T 细胞，并经终浓度 0.5 μg/ml 嘌呤霉素筛选 2 d 后可得到 CD4 阳性表达率约为 55％，CD8 阳性表达率约为 35％的 CAR-T 细胞，且 PRLR-CAR 阳性表达率大于 50％。构建的新型靶向 PRLR 的 CAR-T 细胞可为乳腺癌的免疫治疗提供参考。
Abstract
Chimeric antigen receptor(CAR)T cell therapy has become the focus of research in the field of malignant tumor treatment. Prolactin receptor(PRLR) is one of the potential targets for breast cancer treatment, and its overexpression will affect the occurrence and development of breast cancer. In this study, a three-plasmid system[pHIVgreen fluorescent protein(GFP), psPAX2, pVSVG] was established to package lentivirus and applied to the newly constructed CAR lentiviral expression vector targeting PRLR. By further optimizing the preparation conditions of lentivirus and measuring the titer of lentivirus, CAR-T cells targeting PRLR were constructed successfully. The results showed that when the mass ratio of PRLR-CAR∶psPAX2∶pVSVG was 3∶2∶1, and mixed with transfection reagent at a mass ratio of 1∶2, the condition for the preparation of lentivirus was the optimality, and the titer could reach 1.88× 107 TU/ml after concentration. After infecting human T cells with the lentivirus and screening with a final concentration of 0.5 μg/ml puromycin for two days, CAR-T cells were obtained with the expression positive rates of CD4 and CD8 for 55％ and 35％, respectively. And the expression positive rate of PRLR-CAR could be over 50％. The constructed novel CAR-T cells targeting PRLR can provide a reference for the immunotherapy of breast cancer.
关键词
乳腺癌 /
催乳素受体 /
嵌合抗原受体 /
T淋巴细胞 /
慢病毒
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Key words
breast cancer /
prolactin receptor /
chimeric antigen receptor /
T lymphocyte /
lentivirus
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