人成骨肉瘤顺铂耐药细胞系的建立

目的建立顺铂诱导的人成骨肉瘤多药耐药细胞系(MG63/R)并观察其生物学特性。方法以顺铂为诱导剂,采用大剂量冲击与逐步增加剂量相结合的方法诱导人成骨肉瘤MG63细胞,建立多药耐药系MG63/R。MTT法检测药物敏感性;光镜、透射电镜观察MG63、MG63/R细胞形态及超微结构变化;生长曲线、克隆形成试验检测细胞增殖能力;流式细胞术检测细胞周期、凋亡指数、P-pg、bcl-2、P53蛋白表达。结果经顺铂186d的诱导,建立了MG63/R,其对顺铂的耐药指数为83.557±4.841,对阿霉素、长春新碱、氨甲基蝶呤、环磷酰氨亦产生不同程度的交叉耐药;光镜观察可见MG63/R细胞排列不规则,形态呈三角形、多角形及多核现象;透射电镜显示MG63/R细胞表面突起增加,粗面内质网丰富;细胞周期分析显示细胞增殖能力明显增加而细胞凋亡明显降低;MG63/R细胞P-pg、bcl-2阳性表达较MG63细胞明显增加,P53表达则明显降低。结论本研究建立了稳定的人成骨肉瘤多药耐药细胞系MG63/R,为进一步研究顺铂的多药耐药机制和耐药治疗提供了一种新的实验模型。

Establishment of a Cisplatin-multidrug Resistance Cell Line of Human Osteosarcoma

OBJECTIVE: To establish a multidrug resistance cell line of human osteosarcoma from the MG63 cell line and to test its attributes. METHODS: The human osteosarcoma MG63 cells were exposed to high and gradually increased dose of cisplatin for 186 days to introduce its multidrug resistance (MG63/R). The sensitivity of the multidrug resistance was measured by MTT assay. The morphology and ultramicrostructure of the cells were observed using optical microscopy and transmission electron microscopy. The proliferation ability of the cells was measured by growth curve and colony-forming assay. The cell cycle and apoptosis and the expression of P-pg, bcl-2, P53 in the MG63 and MG63/R cell lines were analyzed by flow cytometry. RESULTS: The resistance index of the MG63/R cells to cisplatin was 83.557 +/- 4.841. The cells also had resistance to doxorubicin, vincristin, methotrexate and cyclophosphamide. Disordered structure of the MG63/R cells was observed through microscopy. The cells appeared in triangle, polygon and polynucleation. The increase of granular endoplasmic reticulums and apophyses was observed through transmission electron microscopy. The proliferation ability of MG63/R increased significantly, with a low apoptosis index. Compared to the MG63, the expressions of P-pg and bcl-2 in the MG63/ R increased while the expression of P53 decreased. CONCLUSION: A multidrug resistance cell line (MG63/R) of human osteosarcoma is established, which will benefit to further studies as a new experimental model.