ABSTRACT
@#[摘 要] 目的：开发基于PiggyBac（PB）转座系统的电转染CAR-T细胞制备方法并鉴定其体外抗肿瘤功能。方法：采用健康人外周血单个核细胞（PBMC）制备T细胞，通过分子克隆技术将CD19基因克隆到PB质粒（转座子）中后经电转染法将转座子和转座酶质粒导入激活的T细胞中，并测定其转染效率，最后运用流式细胞术及荧光素酶发光实验评估其对人Burkitt's淋巴瘤Raji细胞的杀伤能力。结果：电转染制备的CD19 CAR-T细胞转染效率较高（>60%），呈剂量依赖性，且CAR-T细胞相对于Pan-T细胞对Raji细胞杀伤能力显著（P<0.05）。结论：开发的PB转座系统的电转染方法可行，在体外对肿瘤细胞具有显著的杀伤能力，具备临床运用于CD19 CAR-T细胞制备的潜力。
ABSTRACT
@#[摘 要] 目的：探讨shRNA靶向抑制CD38的方法能否增强抗CD38 CAR-T细胞的抗癌功能。方法：构建shRNA靶向抑制CD38的抗CD38 CAR-T细胞的CAR分子，利用逆转录病毒载体包装成功后转导人原代T细胞，制备CAR-T细胞。实验分为shRNA1 CD38 CAR-T组、shRNA2 CD38 CAR-T组和对照组（shR-NC-CD38 CAR-T细胞）。采用qPCR法检测CAR-T细胞CD38 mRNA相对表达水平，计算CAR-T细胞培养0～14 d的增殖倍数，CFSE法检测CAR-T细胞与人Burkitt淋巴瘤细胞Raji-luc或人多发性骨髓瘤外周血B淋巴细胞RPMI-8226-luc共培养时的增殖情况，荧光素酶化学发光法检测CAR-T细胞在不同效靶比（1∶1、1∶2、1∶4、1∶8）时对Raji-luc和RPMI-8226-luc细胞的杀伤效率，ELISA法检测CAR-T细胞杀伤Raji-luc或RPMI-8226-luc细胞时上清液中IFN-γ水平，FCM检测CAR-T细胞表面耗竭T细胞生物标志物PD-1的表达水平。结果：shR-NC-CD38 CAR、shRNA1-CD38 CAR和shRNA2-CD38 CAR逆转录病毒载体的滴度均为1´107拷贝/mL，转导T细胞后，shR-NC-CD38 CAR-T、shRNA1-CD38 CAR-T和shRNA2-CD38 CAR-T细胞的转导效率（CAR的阳性率）分别为60.3%、67.0%和57.4%。与对照组比较，shRNA2-CD38 CAR-T组细胞中CD38 mRNA的表达水平显著降低（P<0.01），显示shRNA-CD38 CAR-T细胞构建成功。shRNA2-CD38 CAR-T组细胞在体外培养增殖能力更强（P<0.05），对2种CD38阳性的肿瘤细胞的杀伤效率更高（均P<0.05）、IFN-γ释放水平更高（均P<0.05）、细胞表面PD-1的表达水平更低（P<0.05）。结论：成功构建一种shRNA靶向抑制CD38的抗CD38 CAR-T细胞，其抗癌功能表现出明显的优势。
ABSTRACT
Background: Endoglucanase, one of three type cellulases, can randomly cleave internal p-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene (JqCel5A) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance.