论文部分内容阅读
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 investigate the cytotoxicity of mDRA-6, an anti-human DR5 monoclonal antibody, on esophageal squamous cell carcinoma EC9706 and 109 cells and its mechanism. Methods: MTT assay was used to detect the cytotoxic effect of mAb mDRA-6 on esophageal cancer cells. Morphological changes of esophageal cancer cells were observed under microscope. DNA fragmentation in esophageal cancer cells was detected by agarose gel electrophoresis. The expression of DR5, apoptosis rate and mitochondrial membrane potential on esophageal cancer cells were detected by flow cytometry. The cytotoxicity of mAb mDRA-6 was assayed by caspase inhibitor. Results: mAb mDRA-6 had significant cytotoxic effect on esophageal squamous carcinoma cells in a dose-and time-dependent manner, but not toxic to NEC cells in esophageal epithelial cells. By mAb mDRA-6 treatment, esophageal squamous cell carcinoma cells showed typical characteristics of apoptosis: cell membrane shrinkage, blistering, chromatin condensation, the formation of apoptotic bodies and DNA fragmentation. The results of flow cytometry showed that the expression of DR5 on the surface of esophageal cancer cells was not observed in NEC cells of esophageal epithelial cells. After 24 hours treatment with 1.2μg / ml mAb mDRA-6, the surface of most EC9706 and 109 cells Both express phosphatidylserine. Inhibitors of Caspase 8 almost completely inhibited mAb mDRA-6-induced apoptosis, with little effect of Caspase 9 inhibitors. In addition, mitochondrial membrane potential did not change in the early stage of esophageal cancer cell apoptosis induced by mAb mDRA-6, and the mitochondrial membrane potential was decreased in the late stage of apoptosis. CONCLUSION: mAb mDRA-6 induces apoptosis primarily through the death receptor signaling pathway and exerts a cytotoxic effect on esophageal squamous cell carcinoma cells. It has a broad range of therapeutic potential in the treatment of tumors with the TRAIL / DR5 system and in the exploration of DR5 functional domains Application prospects.