摘要
目的:探讨抗人DR5功能性单克隆抗体mDRA-6对食管鳞癌EC9706和109细胞的细胞毒作用及其作用机制。方法:利用MTT法检测mAb mDRA-6对食管癌细胞的细胞毒作用;在显微镜下观察食管癌细胞死亡的形态变化;以琼脂糖凝胶电泳检测食管癌细胞中的DNA片段化。利用流式细胞术检测食管癌细胞表面DR5表达、细胞凋亡率和线粒体膜电位;利用caspase抑制剂分析mAb mDRA-6的细胞毒作用机制。结果:mAb mDRA-6对食管鳞癌细胞有显著的细胞毒作用,并呈剂量和时间依赖性,但对食管上皮细胞NEC细胞没有毒性。经mAb mDRA-6处理,食管鳞癌细胞出现典型的细胞凋亡特征:细胞膜皱缩,出泡,染色质浓缩,形成凋亡小体以及DNA片段化等。流式细胞术检测结果显示,食管癌细胞表面表达DR5,而食管上皮细胞NEC细胞不表达;经1.2μg/ml的mAb mDRA-6作用24小时后,大多数食管鳞癌EC9706和109细胞的表面均表达磷脂酰丝氨酸。Caspase 8的抑制剂几乎完全抑制mAb mDRA-6诱导的细胞凋亡,Caspase 9抑制剂的则影响小。此外,在mAb mDRA-6诱导的食管癌细胞凋亡的早期,线粒体膜电位不改变,在凋亡的晚期,线粒体膜电位降低。结论:mAb mDRA-6主要通过死亡受体信号传导途径诱导细胞凋亡,对食管鳞癌细胞产生细胞毒作用,其在以TRAIL/DR5系统进行的肿瘤治疗和探讨DR5功能结构域方面具有广阔的应用前景。
Objective：To study the cytotoxic effect and the mechanisms of a novel anti-human DR5 monoclonal antibody （mDRA-6） in cells of esophageal squamous cell carcinoma（ESCC）.Methods：The cytotoxic effect of mAb mDRA-6 on EC9706 ceils and EC109 cells of ESCC cell lines was determined by MTT assay. The effect of mAb mDRA-6 on the morpha of ESCC cells was observed by fluorencence microscope and DNA fragmentation in ESCC cells was detected by agrose gel electrophoresis. The apoptosis, DR5 expression and mitochondria membrane potential （MMP） of ESCC cells were evaluted by flow cytometry.The cytotoxic mechanisms of mAb mDRA-6 on ESCC ceils was analysed with Caspase inhibitors. Results： mAb mDRA-6 exerted cytotoxicity in EC9706 cells and EC109 cells of ESCC in a dose-dependent and time-dependent manner, but did not in NEC ceils of esophageal epithelial cell line. ESCC cells treated with mDRA-6 exhibited typical apoptostic features in morphology, namely, membrane crenation, bubbling, chromatin condensation, formation of apoptotic bodies, and DNA fragmentation by agrose gel electrophoresis assay.The flow cytometry analysis showed that phosphatidylserine （PS） existed enrichly in majority of ESCC cells treated with 1.2 μg/ml mDRA-6 for 24 hours, and DR5 was expressed on cell surfaces of ESCC, but not NEC cells. Inhibitor of Caspase 8 almost inhibited the apoptosis of ESCC ceils induced by mDRA-6, while that of Caspase 9 hardly did. Moreover, MMP of ESCC cells treated with 1.2 μg/ml mDRA-6 for 12 hours did not change by cytometry assay but almost half of ESCC cells treated with 1.2μg/ml mDRA-6 for 24 hours lest MMP. Conclusion： mDRA-6 may exert cytotoxicity by inducing KSCC cell apoptosis through death receptor signaling pathway, which may be a potential agent in treating ESCC with DR5 as target molecule and exploring the functional domain of DR5.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2008年第6期512-517,共6页
Chinese Journal of Immunology
基金
国家自然科学基金项目(No.30571697)
河南省杰出人才创新基金项目(No.074200510014)
关键词
死亡受体5
TRAIL
细胞凋亡
单克隆抗体
食管鳞癌
Death receptor 5
TRAIL
Apoptosis
Monoclonal antibody
Esophageal squamous cell carcinoma
