中国组织工程研究与临床康复第 15卷第 37期 2011– 09– 10出版Journal of Clinical Rehabilitative Tissue Engineering Research September 10, 2011 Vol.15, No.37 ISSN 1673-8225 CN 21-1539/R CODEN: ZLKHAH6913 1Department of Orthopedics, Ninth People’s Hospital of Chongqing,Chongqing 400700, China; 2Department of Orthopedics,Second Affiliated Hospital ofChongqing Medical University,Chongqing 40010, ChinaChen Yu☆ , Doctor, Attending physician, Department of Orthopedics, Ninth People’s Hospital of Chongqing,Chongqing 400700, Chinachenyu@ Correspondence to: Deng Zhong-liang, Doctor, Professor, Department of Orthopedics, Second Affiliated Hospital of Chongqing Medical University,Chongqing 40010, Chinadeng7586@gmail. comSupported by: the National NaturalScience Foundation of China, No.30772211*Received: 2011-06-10 Accepted: 2011-07-30人骨肉瘤耐阿霉素细胞模型的建立及其生物学特性 *☆陈渝 1,王大勇 1,翁政 1,邓忠良 2Establishment of adriamycin-resistant human osteosarcoma cell line and research on its biological characteristicsChen Yu1, Wang Da-yong1, Weng Zheng1, Deng Zhong-liang2AbstractBACKGROUND: 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 line143B/ADM and to analyze its biological characteristics.METHODS: Increasing concentrations of adriamycin (ADM were applied for 45 days to establish 143B/ADM resistant strain. Thehalf of inhibiting concentrations (IC50 and resistance indexs (RI of different antitumor drugs were measured by CCK assay, andthe cell cycle was analyzed by flow cytometry. The cellular efflux capacity was estimated by rhodamine test. After the cell lineswere treated by ADM, intracellular ADM concentration was detected by fluorospectrophotometer, and the apoptosis wasobserved 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 blotanalysis.RESULTS AND CONCLUSION: After induction for 45 days, the RI of 143B/ADM cells to ADM was 15.7, and the 143B/ADM cellsshowed 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 in143B/ADM cell line (P < 0.01. Flow cytometry and confocal laser microscopy analysis showed that at 72 hours after treatmentwith ADM (10 mg/L, cell apoptosis in 143B/ADM cells was less than that in143B/WT cells. Furthermore, compared with143B/WT, the MDR-1 expression of 143B/ADM was increased significantly with no difference of MRP-1 and LRP expressionamong both cell lines. Multidrug resistance of 143B/ADM cells is related to MDR-1, and has no relationship with MRP-1 and LRP.Chen Y, Wang DY, Weng Z, Deng ZL.Establishment of adriamycin-resistant human osteosarcoma cell line and research on itsbiological characteristics. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(37: 6913-6918.[ ]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 45 d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 G 1和 S 期所占比例增加,而 G 2/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。
关键词:骨肉瘤;阿霉素;多药耐药;多药耐药蛋白 1;细胞模型doi:10.3969/j.issn.1673-8225.2011.37.018陈渝,王大勇,翁政,邓忠良 . 人骨肉瘤耐阿霉素细胞模型的建立及其生物学特性[J].中国组织工程研究与临床康复, 2011, 15(37:6913-6918. [ ]0 引言多药耐药现象是由一种化疗药物诱发,肿瘤组织对该药产生耐药的同时对其他结构和功能无关的化疗药物产生交叉耐药的现象。
多药耐药的发生是影响肿瘤患者化疗效果、预后的主要因素之一 [1-2]。
多药耐药 (multidrug resistance, MDR蛋白 1(MDR-1、多药耐药相关蛋白 1(multidrug resistance-associated protein 1, MRP-1和肺耐药蛋白 (lung resistance protein, LRP是多药耐药基因编码的主要产物。
研究发现, MRP-1、 LRP 和 MDR-1在多种耐药性肿瘤中表达升高,其表达水平与肿瘤耐药性密切相关 [3-5]。
实验拟采取阿霉素 (doxorubicin, ADM为诱导药物,以人骨肉瘤细胞株 143B 为研究对象,采用逐步递增 ADM 浓度间歇作用方法建立人骨肉瘤多药耐药细胞株,并初步探讨其耐药机制。
万方数据陈渝,等 . 人骨肉瘤耐阿霉素细胞模型的建立及其生物学特性P.O. Box 1200, Shenyang 110004 6914www. CRTER .org1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陈渝☆,男, 1977年生,四川省资阳市人,汉族, 2011年重庆医科大学毕业,博士,主治医师,主要从事骨肿瘤耐药机制的研究。