人骨肉瘤耐阿霉素细胞模型的建立及其生物学特性  

Establishment of adriamycin-resistant human osteosarcoma cell line and research on its biological characteristics

作　　者：陈渝[1] 王大勇[1] 翁政[1] 邓忠良[2] 

机构地区：[1]重庆市第九人民医院骨科,重庆市400700 [2]重庆医科大学附属第二医院骨科,重庆市400010

出　　处：《中国组织工程研究与临床康复》2011年第37期6913-6918,共6页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30772211);课题名称:microRNA-1和microRNA-133对骨骼肌卫星细胞增殖分化的调控及机制研究~~

摘　　要：背景:多药耐药是骨肉瘤化疗失败的重要原因,目前其耐药机制不明。目的:诱导建立耐阿霉素的人骨肉瘤细胞株并观察多药耐药蛋白1、多药耐药相关蛋白1和肺耐药蛋白的表达。方法:采用逐步递增阿霉素浓度间歇作用的方法诱导143B/WT细胞株建立143B/阿霉素耐药细胞株。结果与结论:经阿霉素诱导45d建立了143B/阿霉素细胞株,其对阿霉素高度耐药,对顺铂、甲氨蝶呤、异环磷酰胺、长春新碱和紫杉醇亦产生不同程度交叉耐药;流式细胞仪检测显示与143B野生型细胞相比,143B/阿霉素细胞周期中G1和S期所占比例增加,而G2/M期所占比例明显减少;罗丹明外排实验显示,143B/阿霉素细胞药物外排能力显著高于143B/WT细胞(P<0.01);流式细胞仪和激光共聚焦显微镜观察发现,143B/阿霉素细胞阿霉素相关性细胞凋亡率显著低于143B/WT(P<0.01);Western blot检测显示143B/阿霉素细胞多药耐药蛋白1表达水平较143B/WT显著升高(P<0.01),二者多药耐药相关蛋白1和肺耐药蛋白表达差异无显著性意义(P>0.05)。提示143B/阿霉素细胞多药耐药的产生与多药耐药蛋白1表达升高相关。BACKGROUND：Multidrug resistance is a major factor leading to the failure of chemotherapy for human osteosarcoma. However, the precise mechanism remains poorly understood. OBJECTIVE：To establish adriamycin-resistant human osteosarcoma cell line 143B/ADM and to analyze its biological characteristics. METHODS：Increasing concentrations of adriamycin （ADM） were applied for 45 days to establish 143B/ADM resistant strain. The half of inhibiting concentrations （IC50） and resistance indexs （RI） of different antitumor drugs were measured by CCK assay, and the cell cycle was analyzed by flow cytometry. The cellular efflux capacity was estimated by rhodamine test. After the cell lines were treated by ADM, intracellular ADM concentration was detected by fluorospectrophotometer, and the apoptosis was observed by laser scanning confocal microscope and flow cytometry. Meanwhile, the expression of multidrug resistance 1 （MDR-1）, multidrug resistance-associated protein 1 （MRP-1） and lung resistance protein （LRP） were detected by western blot analysis. RESULTS AND CONCLUSION：After induction for 45 days, the RI of 143B/ADM cells to ADM was 15.7, and the 143B/ADM cells showed various resistances to cisplatin, methotrexate, isophosphamide, vincristine and taxinol. Compared with 143B/WT cell line, there were less cells in G2/M phase and more cells in G1 and S phases, markedly decreased intracellular rho123 and ADM in 143B/ADM cell line （P 0.01）. Flow cytometry and confocal laser microscopy analysis showed that at 72 hours after treatment with ADM （10 mg/L）, cell apoptosis in 143B/ADM cells was less than that in 143B/WT cells. Furthermore, compared with 143B/WT, the MDR-1 expression of 143B/ADM was increased significantly with no difference of MRP-1 and LRP expression among both cell lines. Multidrug resistance of 143B/ADM cells is related to MDR-1, and has no relationship with MRP-1 and LRP.

关 键 词：骨肉瘤 阿霉素 多药耐药 多药耐药蛋白1 细胞模型 

分 类 号：R318[医药卫生—生物医学工程]