人乳腺癌细胞系MCF-7/W和MCF-7/ADM细胞中阿霉素结合蛋白的分析  

The difference of ADM binding protein in human breast cancer cell line MCF-7/Wild and MCF-7/ADM Cell

作　　者：王妍 张莲芬 冯磊 金坚 

机构地区：[1]江南大学医药学院分子药理实验室,江苏无锡214036

出　　处：《中国药理学通报》2007年第9期1188-1193,共6页Chinese Pharmacological Bulletin

基　　金：上海市科学技术委员会生物医药重大科技攻关项目(No060219020)

摘　　要：目的通过比较野生型和耐药型人乳腺癌细胞系MCF-7中阿霉素(ADM)结合蛋白的差异,探讨耐药型MCF-7的多药耐药机制。方法酯化脱水法制备生物素化ADM,HPLC纯化,MS分析确证。以ADM耐药指数≥200的MCF-7细胞(MCF-7/ADM)和野生型MCF-7细胞(MCF-7/W)为研究材料,采用链亲和素-亲和甄别磁珠法从两种细胞全蛋白中分离出与ADM结合的蛋白。经SDS-PAGE,肽质谱指纹图谱(MALDI_TOF_MS)分析ADM结合蛋白的种类。细胞迁移实验比较MCF-7/W和MCF-7/ADM细胞的运动能力。结果生物素化ADM、ADM对MCF-7/W的IC50分别为0.796μmol.L-1和0.547μmol.L-1。分别从MCF-7/W和MCF-7/ADM中采用亲和甄别磁珠法获得ADM结合蛋白20种及17种,SDS-PAGE分析两者有一条差异条带,序列分析共同蛋白为myosin,beta-actin,alpha-actin,keratin。MCF-7/W和MCF-7/ADM细胞在培养72h后,前者的迁移速度较快。结论除了因方法学灵敏度限制未能解析的结合蛋白外,MCF-7细胞内的主要ADM结合蛋白是细胞骨架蛋白和参与细胞运动的蛋白。ADM与MCF-7细胞中的骨架蛋白结合后,对细胞的迁移运动有一定的影响作用。Aim To study the mutidrugresistance mechanism of tumor cell by comparing the binding pro-tein with Adriamycin（ADM）in the MCF-7/Wild cell and MCF-7/ADM resistance cell.Methods The biotin-ADM compound was synthesized with the esterification-dehydration method,purified with HPLC and analyzed with mass spectrograph.The binding proteins against Adriamycin from MCF-7/ADM or MCF-7/Wild cell whose resistance index is above 200 were obtained respectively by the affinity screening magnetism bead method with biotin-ADM compound as a specific ligand.Some binding proteins with Adriamycin were identified with the MALDI-TOF-MS after isolating them with SDS-PAGE.Cell motor ability was compared in the migration experiment.Results The variability of biotin-ADM and ADM was negligible,while that IC50 of the two compounds to MCF-7/Wild was 0.796 μmol·L^-1nd 0.547 μmol·L^-1espectively.The 20 types binding proteins with ADM were found from MCF-7/Wild cell,and 17 types binding proteins from MCF-7/ADM resistance cell.There was one protein band difference from the two cells on SDS-PAGE with silver staining.The same binding proteins on the two cells that identified with MALDI-TOF-MS were myosin,beta-actin,alpha-actin,keratin.MCF-7/W and MCF-7/ADM both migrated after being cultivated 72 h,with a faster migration of MCF-7/ADM.Conclusion There are different binding protein with ADM on the MCF-7/Wild cell and MCF-7/ADM resistance cell.The same binding protein with ADM on the two cells belongs to the cystoskeleton protein.The binding of the ADM with the cell cystoskeleton protein has some effect to the migration of the MCF-7/ADM.

关 键 词：MCF-7 阿霉素 生物素化阿霉素 亲和甄别磁珠细胞迁移 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R329.25[医药卫生—基础医学]