抗CD71人-鼠嵌合抗体的制备及其诱导肿瘤细胞凋亡

The Preparation of Anti-CD71 Mouse/Human Chimeric Antibody and Its Induced Apoptosis of Tumor

目的 :研究在液氮中冻存 2年的转染瘤细胞株 (D2C)复苏后其分泌抗CD71人 -鼠嵌合抗体的特异性、分泌量及诱导肿瘤细胞凋亡的效应。方法 :用ELISA方法检测转染瘤细胞培养上清的抗体分泌量。经DEAE SephadexA 5 0离子交换法纯化产生的抗体 ,并采用SDS PAGE电泳鉴定 ;台盼蓝拒染法观察其对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。结果 :Balb/c裸鼠腹腔接种转染瘤细胞后 ,每只裸鼠腹水量在 3～ 5mL。抗体经纯化 ,产量约为 1～ 2mg/mL腹水。经SDS PAGE电泳鉴定 ,在分子量 5 5kD和 2 7kD处 ,可见有抗体IgG重链和轻链的染色条带。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 ,且二者抑制作用无显著性差异 (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。结论 :液氮中冻存 2年的转染瘤细胞体外生长良好 ,抗体分泌稳定 ,特异性高 ,并可诱导K5 6

Objective:To study the specificity, productivity and tumor-induced apoptosis of an anti-CD71 mouse/human chimeric antibody (D2C)secreted by transfectomas kept in liguid nitrogen for tow years.Methods: Chimeric Ab production in culture supernatant was measured by ELISA. The antibodies from ascites were purified by DEAE-Sephredax-A50 ion-exchange chromatography and confiremed by SDS-PAGE. Cell survival fraction was determined using the trypan blue dye exclusion assay. Cell proliferation test and MTT assay were used to determine effects of antibody(D2C) and murine McAb on K562 cells. Apoptosis was detected by DNA gel electrophoresis and transmission electron microscope.Results: After inoculation of antibody-secreted transfectomas in abdominal cavity of Balb/c, 3 ～5 mL ascites was abtained from each nude mouse and purified antibody(D2C) 1～2 mg per millilitre ascites. The SDS-PAGE electrophoresis of purified antibody(D2C) indicated two protein bands of 55kDa and 25kDa. Antibody(D2C) can inhibit proliferation of K562 cells, and its effect of inhibition was no difference(P< 0.05) with murine McAb. Typical DNA ladder and apoptosis form of K562 cells induced by antibody(D2C) showed is observed by electrophoresis and transmission electron microscope, respectively.Conclusions:The antibody(D2C) kept in liguid nitrogen for tow years production from the transfectomas is stable. Antibody(D2C) and murine McAb can induce K562 apoptosis.