钙网织蛋白在阿霉素诱导的早期凋亡A549细胞免疫原性中的作用  

The Role of Calreticulin in the Immunogenicity of Early Apoptotic Human Lung Cancer Cells A549 Induced by Doxorubicin

作　　者：窦征岳[1] 江其生[2] 何蕊[2] 宋秀军[2] 李峰生[2] 

机构地区：[1]保定市第三医院,河北保定071000 [2]第二炮兵总医院,北京100088

出　　处：《肿瘤学杂志》2014年第9期730-734,共5页Journal of Chinese Oncology

摘　　要：[目的]研究钙网织蛋白在阿霉素诱导的凋亡人肺癌A549细胞免疫原性中的作用。[方法]体外培养人肺癌A549细胞,将阿霉素加入A549细胞培养液中,使其终浓度分别为1×10-3g/L、3×10-3g/L、5×10-3g/L,12 h后以吖啶橙/溴化乙锭(AO/EB)双染法检测细胞凋亡;应用激光共聚焦显微镜观察凋亡A549细胞表面钙网织蛋白暴露情况。阿霉素3×10-3g/L作用12 h后诱导的凋亡细胞为抗原组(A组),A组加入钙网织蛋白阻断蛋白(A-Ca组),分别刺激外周血单个核细胞,72h后以MTT法检测其对培养的A549细胞的杀伤效果。[结果]阿霉素3×10-3g/L终浓度作用A549细胞12h后,凋亡率为62.93%±1.70%。经激光共聚焦显微镜检测凋亡A549细胞表面可见钙网织蛋白抗体荧光。外周血单个核细胞杀伤实验MTT检测OD值:A-Ca组0.537±0.056,A组0.437±0.023(P<0.05)。[结论]阿霉素诱导凋亡的A549细胞表面有钙网织蛋白的暴露。阿霉素诱导的凋亡A549细胞经添加钙网织蛋白阻断蛋白后,其免疫原性下降,表明钙网织蛋白在其免疫原性中起重要作用。[Purpose] To investigate the role of calreticulin in the immunogenicity of apoptotic human lung cancer cells A549 induced by doxorubicin. [Methods] A549 cells were cultured in medium containing doxorubicin （1×10^-3g/L,3×10^-3g/L,5×10^-3g/L） in vitro. After 12h,apoptosis of A549 cells was detected by acridine orange/ethidium bromide （AO/EB） double staining;Calreticulin on the surface of apoptotic A549 cells was detected by laser scanning confocal microscope. Peripheral blood mononuclear cells were stimulated by apoptotic cells （group A） and apoptotic cells plus ealreticulin blocking protein （group A-Ca ） respectively.The killing effect on A549 cells was evaluated by MTT method after 72h. [Resuh] Apoptotic rate of A549 cells cultured with doxorubicin （3×10^-3g/L）was 62.93% ±1.70% and calreticulin was detected on the surface of A549 cells.Optical density of group A-Ca and group A was 0.537±0.056 and 0.437± 0.023 （P〈0.05） respectively by MTT method. [Conclusion] Calreticulin might be found on the surface of apoptotic A549 cells induced by doxorubiein. Calreticulin blocking protein reduces the immunogenicity of apoptotic A549 cells. It suggests that calreticulin seems to play an important role in the immunogenicity of apototic A549 cells.

关 键 词：肺肿瘤 细胞凋亡 阿霉素 免疫原性 钙网织蛋白 

分 类 号：R730.3[医药卫生—肿瘤]