人骨肉瘤多药耐药细胞模型建立及生物学形状分析

Establishment of Multidrug-resistant Human Osteosarcoma Subclones

目的 :建立人骨肉瘤细胞多药耐药 (MDR)细胞亚系。方法 :采用多柔比星间断冲击法诱导骨肉瘤细胞 ,并用免疫荧光法检测P 糖蛋白 (P gp)的表达、四唑蓝 (MTT)比色法对MDR表型鉴定和多柔比星结合实验法检测肿瘤细胞耐药性及耐药逆转药。结果 :该实验建立 6株MDR细胞亚系MG 63 R1～ 6 ,免疫荧光术可以检测到P gp的表达 ,MTT比色法、多柔比星结合试验方法显示各亚系细胞的多药耐药性明显增加 ,并且维拉帕米可以拮抗P gp的作用。结论 :Mdr 1 P gp在MDR的特性上起着至关重要的作用 ,这些骨肉瘤MDR细胞亚系模型为进一步研究骨肉瘤耐药特征及逆转方法奠定基础。

Objective:To establish multidrug-resistant(MDR) subclones of human osteosarcoma. Methods:The human osteosarcoma cell line MDR was subjected to a series of short-time pulse exposures to doxorubicin in 6 increasing concentrations beginning from 0.01 to 4 μg·mL -1.The procedure was repeated over and over again and the experiment lasted 5 months.The phenotype of the subclones was analyzed with MTT colorimetry. P-glycoprotein(P-gp) was detected with immunofluorscence. Drug resistance of tumor cells and drug resistance reversing agents were tested with the doxorubicin-binding assay(ABA). Results:6 subclones of the MDR cell line, MG63/R 1-6, were established.P-gp expression on these cells was demonstrated by immunofluorscence. MTT colorimetry and ABA revealed that the multidrug-resistance of all 6 subclones was strikingly stronger than that of the parent cell line. Besides, verapamil was shown to antagonize the effect of P-gp. Conclusion:Expression of Mdr-1/P-gp was shown to be the key factor to regulate the MDR phenotype of osteosarcoma. These newly described multidrug-resistant osteosarcoma subclones are useful models for further study of the features of drug resistance of osteosarcoma and for the development of methods to reverse the drug resistance.