EGFRvⅢ胞外段修饰DC诱导CTL对EGFRvⅢ^(+)胃癌细胞的杀伤作用  

EGFRvⅢ extracellular segment modification of DC induced the killing effect of CTL on EGFRvⅢ^(+) gastric cancer cells

作　　者：侯皓凡 夏兴洲[1] 李金丽 王万聪 李冲慧 姚倩 周潇潇 HOU Haofan;XIA Xingzhou;LI Jinli;WANG Wancong;LI Chonghui;YAO Qian;ZHOU Xiaoxiao(Department of Gastroenterology,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]郑州大学第五附属医院消化内科,河南郑州450000

出　　处：《胃肠病学和肝病学杂志》2022年第7期798-801,811,共5页Chinese Journal of Gastroenterology and Hepatology

基　　金：河南省科技攻关计划(162102310513)。

摘　　要：目的构建表达EGFRvⅢ的树突状细胞(dendritic cells,DC),并使其诱导细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)成熟,检测该细胞对胃癌细胞及EGFRvⅢ^(+)胃癌细胞的杀伤作用。方法制备携带EGFRvⅢ的慢病毒,并用荧光定量法测定慢病毒滴度。分别使用携带EGFRvⅢ的慢病毒、空白对照的慢病毒转染DC,Western blotting测定EGFRvⅢ的表达。自身CTL分别与EGFRvⅢ^(+)DC、空白慢病毒感染的DC、成熟DC共培养,采用ELISA法检测上清液中IL-10和IFN-γ水平。按不同比例(1∶5、1∶10、1∶20、1∶40)将传代后的SGC7901细胞和SGC7901-EGFRvⅢ^(+)细胞分别加入不同组的CTL培养24 h,MTT法测定CTL对胃癌细胞的杀伤作用。以上所有实验均重复3次。结果慢病毒感染DC的感染率可达58%。EGFRvⅢ将慢病毒作为载体可使DC感染并成功表达EGFRvⅢ。在体外应用EGFRvⅢ^(+)DC诱导CTL成熟,可使CTL产生更多IL-10、INF-γ,且呈剂量依赖性(P<0.01),其对胃癌细胞的杀伤作用更强(P<0.05),而且对高表达EGFRvⅢ的胃癌细胞的杀伤作用最强。结论相同条件下,由EGFRvⅢ^(+)DC诱导成熟的CTL对EGFRvⅢ^(+)胃癌7901细胞在体外的免疫杀伤作用明显增强。Objective To construct dendritic cells(DC)expressing EGFRvⅢ,make it induce the cytotoxic T lymphocyte(CTL)to mature,and to detect the killing effect of the CTL on gastric cancer cells and EGFRvⅢ^(+)gastric cancer cells.Methods The lentivirus carrying EGFRvⅢ was prepared and the titer of lentivirus was determined by fluorescence quantitative method.DC was transfected with a lentivirus carrying EGFRvⅢ and a control lentivirus,respectively,and the expression of EGFRV was determined by Western blotting.Autologous CTL were co-cultured with EGFRvⅢ^(+)DC,blank lentivirus-infected DC,and mature DC,respectively.The levels of IL-10 and IFN-γ in the supernatant were detected by ELISA.SGC7901 cells and SGC7901-EGFRvⅢ^(+)cells were added into different groups of CTL in different proportions(1∶5,1∶10,1∶20,1∶40)and cultured for 24 hours.The killing effect of CTL on gastric cancer cells was determined by MTT assay.All the above experiments were repeated for 3 times.Results The infection rate of DC infected by lentivirus reached 58%.EGFRvⅢ DC could be infected with lentivirus as a vector and successfully express EGFRvⅢ.When EGFRvⅢ^(+)DC was used in vitro to induce CTL to mature,CTL could produce more IL-10 and INF-γ in a dose-dependent manner(P<0.01).The killing effect of EGFRvⅢ on gastric cancer cells was stronger(P<0.05),and the killing effect of EGFRvⅢ on EGFRvⅢ^(+)gastric cancer cells was the strongest.Conclusion Under the same condition,the mature CTL induced by EGFRvⅢ^(+)DC significantly enhanced the immune killing effect against EGFRvⅢ^(+)gastric cancer 7901 cells in vitro.

关 键 词：慢病毒 EGFRvⅢ 胃癌7901细胞 树突状细胞 细胞因子 

分 类 号：R735.2[医药卫生—肿瘤]