耐阿霉素人骨肉瘤细胞株的建立及其耐药机制探讨

Establishment of an adriamycin resistance cell line of human osteosarcoma and study of its resistant mechanisms

目的诱导并建立耐阿霉素(ADM)的人骨肉瘤细胞株并探讨其耐药机制。方法采用逐步增加药物剂量冲击诱导方法诱导人成骨肉瘤原代Saos-2细胞株;MTT法检测原代与耐药细胞株对ADM、顺铂(DDP)、甲氨蝶呤(MTX)、异环磷酰胺(IFO)、表柔比星(EPI)、比柔比星(THP)、紫杉醇(Paclitaxel,PTX)药物敏感性;利用光学显微镜、透射电镜观察细胞形态及超微结构变化;RT-PCR和IHC法分别检测多药耐药基因1(MDR1)、多药耐药相关蛋白(MRP)基因及其相应蛋白(P-gp)、MRP的表达。结果经167d的诱导,建立Saos-2/ADM1、Saos-2/ADM4细胞株,其对ADM的耐药指数分别为原代细胞株的49.8和74.6倍;耐药细胞株对MTX、EPI、THP、PTX亦产生不同程度的交叉耐药(P<0.05),对DDP仍然敏感(P>0.05);光镜观察Saos-2/ADM1、Saos-2/ADM4细胞株细胞体积增大,多核现象较原代Saos-2细胞株明显增加;透射电镜显示Saos-2/ADM1、Saos-2/ADM4细胞株表面突起较原代Saos-2细胞株减少、且核仁增大增多;细胞生长曲线显示耐药细胞株增殖能力下降。MDR1mRNA、MRPmRNA和P-gp、MRP在各耐药细胞株表达阳性。结论MDR1 mRNA、MRP mRNA及其相应蛋白参与了耐药细胞株耐药的形成,这些骨肉瘤耐药细胞株为进一步研究骨肉瘤耐药特征及逆转方法打下了基础。

Aim To establish a resistant human osteosarcoma cell line （ Saos-2/ADM 1 and Saos-2/ADM4 ） from the Saos-2 cell line and study its resistant mechanisms. Methods Saos-2 cells were pulse exposed in gradually increased dose of ADM culture medium. The sensitivity of the resistance drug from the Saos-2, Saos-2/ADM1 and Saos-2/ADM4 cell lines to ADM,DDP,EPI, MTX ,THP and PTX was measured by MTI＇ assay. The morpholo- gy and ultramicro structure of the cell lines were observed by optical microscopy and transmission electronic microscopy. The expression level of MDR1 mRNA, MRP mRNA and their proteins P-gp, MRP was examined by reverse transcription polymerase chain reaction and immunohistochemistry. Results Resistance cell lines were established after 167 days. The resistance index to methotrexate of the Saos-2/ADMland Saos-2/ADM4 cell lines to ADM was 49. 8 and 74.6 times than that of Saos-2 respectively. The two cell lines had resistance to MTX, IFO, EPI ,THP and PTX （P 〈0. 05）. The cell was found to remain sensitive to cis- platin （P 〉 0. 05 ）. Disordered structure of the Saos-2/ADMland Saos-2/ADM4 cells was observed through microsco- py. The cells appeared in coenocytic. The decrease of cell villus and the increase of nucleoli were observed through transmission electronic microscopy. The proliferation ability of Saos-2/ ADMland Saos-2/ADM4 cells decreased significantly. MDR1 mRNA,MRP mRNA,P-gp and MRP showed positive staining in resistance cell lines. Conclusion The genes of MDR1 mRNA, MRP mRNA and their corresponding proteins participated in the formation of muhidrug resistance in resistant adriamycin cell line. These newly-described resistant osteosarcoma cell lines were useful models for further characterization of drug resistance in osteosarcoma and for the development of treatment protocol.