潜伏膜蛋白2A嵌合抗原受体-T细胞的制备及对鼻咽癌细胞杀伤作用的研究  

Construction of latent membrane protein 2A chimeric antigen receptor-T cells and their lethal effects on nasopharyngeal carcinoma cells

作　　者：陈渊[1] 陈仁杰[1] 黄骁辰 汤根兄[1] 蒯兴旺 章明炯 张大为[1] 唐奇 朱进[1] 冯振卿[1] Chen Yuan;Chen Renjie;Huang Xiaochen;Tang Genxiong;Kuai Xingwang;Zhang Mingjiong;Zhang Dawei;Tang Qi;Zhu Jin;Feng Zhenqing(Department of Otorhinolaryngology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing 200031, China)

机构地区：[1]南京医科大学第二附属医院耳鼻咽喉头颈外科,200031

出　　处：《中华耳鼻咽喉头颈外科杂志》2018年第12期925-930,共6页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

摘　　要：目的制备靶向潜伏膜蛋白2A(latent membrane protein2A,LMP2A)的嵌合抗原受体(chimeric antigen receptor,CAR)-T细胞,观察LMP2ACAR-T细胞体内外对鼻咽癌细胞的杀伤作用。方法2016年9月至2017年12月,本研究以基因工程技术构建抗LMP2A慢病毒表达载体,测序鉴定并通过Western blot法验证抗LMP2ACAR在293T细胞中的表达;通过质粒包装体系制备LMP2ACAR慢病毒,并感染人T细胞,制备LMP2ACAR-T细胞;CCK-8法检测LMP2ACAR-T细胞对鼻咽癌细胞的杀伤作用。酶联免疫吸附试验(ELISA)法检测LMP2A抗原激活后的LMP2ACAR-T细胞白细胞介素(IL)-2与干扰素(IFN)-γ的分泌水平。体内实验观察LMP2ACAR-T细胞对鼻咽癌移植瘤的抑瘤作用。SPSS21.0统计学软件用于统计分析。结果PCR结果显示抗LMP2ACAR片段大小约为1500bp,与理论值相一致,测序显示序列正确;Westernblot结果显示抗LMP2A慢病毒表达载体能在293T细胞中表达;CCK-8法结果发现在效靶比为20∶1、10∶1与5∶1时,LMP2ACAR-T细胞对LV-LMP2A-CNE1细胞的杀伤率分别为(72.11±9.75)%、(54.65±5.42)%与(36.68±3.80)%,与CD19CAR-T细胞、T细胞相比差异有统计学意义(P<0.05),而对CNE1细胞的杀伤作用与CD19CAR-T细胞和T细胞相比差异无统计学意义;ELISA法结果发现LMP2ACAR-T细胞与LV-LMP2A-CNE1细胞共培养上清中IL-2与IFN-γ的含量,显著高于与CNE1细胞共培养上清,差异具有统计学意义(P<0.05);体内实验中LMP2ACAR-T细胞组瘤体体积为(80.3±10.0)mm3,显著小于各对照组,差异具有统计学意义(P<0.05)。结论成功制备的LMP2ACAR-T细胞对LMP2A阳性的鼻咽癌细胞具有显著靶向杀伤效应。Objective To produce latent membrane protein 2A (LMP2A) chimeric antigen receptor (CAR)-T cells and detect the lethal effect of LMP2A CAR-T cells on nasopharyngeal carcinoma (NPC) cells. Methods The study was conducted from September 2016 to December 2017.Genetic engineering technology was used to construct anti-LMP2A CAR lentiviral expression vector and sequencing was identified. The expression of anti-LMP2A CAR in the 293T cells was confirmed by western blot. CCK8 assay was used to evaluate the cytotoxicity of LMP2A CAR-T cells to NPC cells. ELISA assay was performed to test IL-2 and IFN-γ releasing of activated LMP2A CAR-T cells. The inhibition effect of LMP2A CAR-T cells on NPC xenograft tumor was observed in vivo. Statistical analysis was performed by statistical software SPSS 21.0. Results The results of PCR and sequencing showed that anti-LMP2A CAR lentiviral expression vector was constructed successfully. The result of western blot indicated the expression of anti-LMP2A CAR in the 293T cells effectively. The results of CCK-8 assay showed that the killing activities of LMP2A CAR-T cells to LV-LMP2A-CNE1 cells were (72.11±9.75)%, (54.65 ±5.42)% and (36.68±3.80)% at 20∶1, 10∶1 and 5∶1 ratio of effective cells to target cells, and had a statistical difference compared to CD19 CAR-T cells and T cells (P<0.05). There was no significant difference in the killing activities of LMP2A CAR-T cells to CNE1 cells compared with CD19 CAR-T cells and T cells. The results of ELISA showed that the content of IL-2 and IFN-γ in the co-culture supernatant of LMP2A CAR-T cells and LV-LMP2A-CNE1 cells was significantly higher than that of LMP2A CAR-T cells and CNE1 cells which had statistical difference (P<0.05);In vivo experiment, the volume of LMP2A CAR-T cell group was (80.3±10.0) mm3 which was significantly lower than that of the control groups, and the difference was statistically significant (P<0.05). Conclusion LMP2A CAR-T cells are successfully prepared and have an obvious targeting cytotoxicity on LMP2A-pos

关 键 词：293T细胞 细胞杀伤作用 潜伏膜蛋白2A 鼻咽癌细胞 抗原受体 制备 WESTERNBLOT 嵌合 

分 类 号：R739.63[医药卫生—肿瘤]