抗CD71人-鼠嵌合抗体诱导K562细胞凋亡及其机制探讨

The anti-CD71 mouse/human chimeric antibody induced apoptosis of K562 and its molecular mechanisms

目的 研究抗CD71人 鼠嵌合抗体诱导K5 6 2细胞凋亡的效应及其机制。方法 台盼蓝拒染法观察抗CD71人 鼠嵌合抗体对K5 6 2细胞生存的影响 ;细胞计数和MTT试验测定嵌合抗体及鼠源性抗体对K5 6 2细胞的作用 ;琼脂糖凝胶电泳和透射电镜观察K5 6 2细胞凋亡 ,以AnnexinⅤ检测其凋亡率及进行细胞周期分析 ;并检测胞浆中细胞色素C和活化的caspase 8的相对含量。结果 抗CD71嵌合抗体及鼠源性抗CD71抗体均可抑制K5 6 2细胞的增殖作用 ,且二者抑制作用差异无显著性(P >0 .0 5 )。经琼脂糖凝胶电泳和透射电镜观察分别可见抗体诱导的K5 6 2细胞DNA梯形条带和细胞凋亡的形态改变 ,细胞周期分析可见其将细胞周期阻止在G1 期 ,胞浆中细胞色素C含量增加而活化的caspase 8的含量不增加。结论 抗CD71人 鼠嵌合抗体可通过线粒体死亡途径诱导K5 6 2人红白血病细胞凋亡 ,其诱导凋亡可能与其干扰细胞铁离子转运有关。

Objective To study the effect and mechanism of the tumor apoptosis induced by the anti-CD71 mouse/human chimeric antibody (D2C). Methods The effect of the chimeric antibody on the K562 cell survival was determined by the trypan blue dye exclusion assay. Cell proliferation test and MTT assay were used to determine the effects of antibody(D2C) and murine McAb on K562 cells. Apoptosis was tested by DNA gel electrophoresis and investigated by transmission electron microscopy. Apoptosis rate and cell cycle were detected by Annexin Ⅴ and the relative content of cytochrome C and activated caspase-8 in cytoplasm were tested. Results Both CD71 chimeric antibody and murine CD71 antibody were able to inhibit proliferation of K562 cells, and their inhibitions were of no difference(P<0.05). Typical DNA ladder and apoptosis of K562 cells induced by the antibody(D2C) were observed by electrophoresis and transmission electron microscopy, respectively. The cell cycle was blocked at G 1 phase, the content of cytochrome C increased and the activated caspase-8 remained at the lower level in cytoplasm. Conclusion Antibody(D2C) and murine McAb can induce K562 apoptosis by the pathway of mitochondrial death, the induction of apoptosis would be related with interfering iron transportation in cells.