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食管癌特异性PTK7-CAR-T细胞的制备及其功能研究
作者:胡欣1  肖婷2  陈思林1  李婷婷1  冯旭琴1  李彬3  刘康2 
单位:1. 南充市中心医院/川北医学院第二临床医学院 肿瘤中心, 四川 南充 637000;
2. 南充市中心医院/川北医学院第二临床医学院 组织与工程干细胞研究所 四川 南充 637000;
3. 川北医学院 临床医学系, 四川 南充 637000
关键词:食管癌 蛋白酪氨酸激酶-7 嵌合抗原受体-T淋巴细胞 增殖活性 特异杀伤率 
分类号:R735.1
出版年·卷·期(页码):2022·50·第九期(1089-1095)
摘要:

目的:制备食管癌特异性蛋白酪氨酸激酶7(PTK7)-嵌合抗原受体(CAR)-T淋巴细胞(PTK7-CAR-T),并研究其功能。方法:以抗PTK7单链抗体为靶向分子构建CAR慢病毒表达质粒,包装制备慢病毒颗粒lenti-CAR,共同培养并转染T细胞,获得PTK7-CAR-T细胞,鉴定重组慢病毒表达载体,同时构建Control-CAR-T细胞。采用流式细胞术检测各CAR-T细胞增殖活性;靶细胞杀伤实验检测特异杀伤率;酶联免疫吸附试验(ELISA)检测不同CAR-T细胞因子释放能力。结果:成功构建PTK7-CAR-T细胞和Control-CAR-T细胞;与Control-CAR+TE5组、Control-CAR+TE9组相比,嵌合PTK7抗原的CAR-T细胞可以特异性识别TE5、TE9细胞并降低其增殖活性(P<0.05),同时提高自身的杀伤能力和细胞因子释放能力(P<0.05),且其杀伤能力随效靶比的升高而增强(P<0.05);与Control-CAR+HEEC组相比,嵌合PTK7抗原的CAR-T细胞对HEEC增殖活性无影响(P>0.05),且不会影响其自身的特异杀伤率和细胞因子释放能力(P>0.05)。结论:PTK7-CAR-T细胞可特异性识别人食管鳞癌细胞并降低其增殖活性,提高自身的特异杀伤能力和细胞因子释放能力,起到抗癌的效果。

Objective: To prepare esophageal cancer specific protein tyrosine kinase 7(PTK7)-chimeric antigen receptor(CAR)-T lymphocytes(PTK7-CAR-T) and study their functions. Methods: The CAR lentiviral expression plasmid was constructed with the anti-PTK7 single-chain antibody as the targeting molecule, the lentivirus particles lenti-CAR was packaged, and co-cultured and transfected T cells to obtain PTK7-CAR-T cells, and the recombinant lentiviral expression vector was identified. Control-CAR-T cells were constructed at the same time. The proliferative activity of CAR-T cells was detected by flow cytometry. The specific killing rate was detected by target cell killing assay. Enzyme-linked immunosorbent assay(ELISA) was used to detect the release ability of different CAR-T cytokines. Results: PTK7-CAR-T cells and Control-CAR-T cells were successfully constructed. Compared with Control-CAR+TE5 group and Control-CAR+TE9 group, CAR-T cells chimeric with PTK7 antigen could specifically recognize TE5 and TE9 cells and reduce their proliferation activity(P<0.05), while improving their own specific killing ability and cytokine release ability(P<0.05), and its killing ability increased with the increase of effective target ratio(P<0.05). Compared with Control-CAR+HEEC group, CAR-T cells chimeric with PTK7 antigen had no effect on the proliferation activity of HEEC(P>0.05), and it didnt affect their own specific killing rate and cytokine release ability(P>0.05). Conclusion: PTK7-CAR-T cells can specifically recognize human esophageal squamous cell carcinoma cells and reduce their proliferation activity, improve their specific killing ability and cytokine release ability, and play an anti-cancer effect.

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