人骨肉瘤耐药细胞系MG-63/PTX的建立及多药耐药机制研究

A study of establishment of multidrug resistant cell line MG-63/PTX in human osteosarcoma and the mechanism of multidrug resistance

目的建立人骨肉瘤细胞多药耐药亚系,探讨骨肉瘤细胞多药耐药的发生机制。方法采用紫杉醇大剂量间断冲击培养法,诱导建立紫杉醇耐药骨肉瘤细胞系(MG-63/PTX),并运用MTT法检测6种药物敏感性变化及细胞生长规律变化,流式细胞仪分析亲本细胞系和耐药细胞系的细胞膜表面P-gp蛋白表达率、罗丹明123排出情况,RT-PCR检测细胞内耐药基因的变化,免疫细胞化学法检测细胞内P-gp蛋白的表达,Westernblotting法检测细胞内P-gp蛋白的表达。结果历时12个月建成人骨肉瘤耐药细胞系MG-63/PTX,能在含PTX1μg/mL的培养基中稳定生长并传代,其对PTX的耐药指数为69.8,并与卡铂、顺铂、表柔比星、甲氨蝶呤、阿霉素等多种化疗药物存在不同程度的交叉耐药性;罗丹明123排出实验显示MG-63/PTX内罗丹明含量明显低于亲本细胞;RT-PCR结果显示在MG-63/PTX细胞系中存在MDR1、MRP1、LRP、ABCG2基因的表达,而在MG-63细胞系中无MDR1基因的表达。免疫细胞化学、流式细胞仪及Western blotting实验均显示在MG-63/PTX中存在P-gp的表达。结论 MDR1/P-gp在多药耐药的特性上起着至关重要的作用,而且骨肉瘤多药耐药细胞亚系为进一步研究骨肉瘤耐药特征及逆转方法打下基础。

Objective To explore the pathogenesis of multidrug resistance of osteosarcoma through establishing multidrng-resistant cell line subpopulation of human osteosarcoma. Methods Paclitaxel discontinuous impact culture method of high-dose was conducted to induce the development of paclitaxel drug-resistant cell line MG-63/PTX of osteosarcoma. 3- （4,5-Dimethylthiazol-2-yl） -2,5-diphenyltetrazolium bromide （MTT） was used to test the change of drug susceptibility and law of cell growth of 6 medications. Flow cytometry was used to analyze the expression rate of human permeability glycoprotein （P-gp） on the surface of cell membrane of parent cell line and drug-resistant cell line and the eduction of rhodamine123. Reverse transcription-polymerase chain reaction （RT-PCR） was used to exam the change of drug-resistant gene in cells. Immunocytochemistry was applied to exam the expression of P-gp in cells. Western blotting was conducted to exam the expression of P-gp in cells. Results Drug-resistant cell line MG-63/PTX of human osteosarcoma was successfully established after 12 month, and it could grow stably and go down to the future generation in the culture medium with 1ug / ml plataxel. Regarding plataxel, the drug-resistant index of cell line MG-63/PTX was 69.8, and it exhibited cross-resistance to many other chemotherapeutic agents such as carboplatin, cisplatin, epirubicin, methotrexate and adriamycin etc to different degrees. From the eduction of rhodamine, it was showed that the content of rhodamine in cell line MG-63/PTX was obvious lower than the parent cell line. The results of RT-PCR showed that the expressions of MDR1, MRP1, LRP and ABCG2 exists in the cell line MG-63/ PTX. MDR1 was not expressed in cell line MG-63. P-gp expression existed in cell line MG-63/PTX through the test of immunohistochemical, flow cytometry and western blotting. Conclusions MDR1/P-gp may play a crucial role in the characteristics of multidrug resistance. Multidrug-resistant cell line subselies in human osteosarcoma provide the basis for future research of characteristics of multidrug resistance in osteosarcoma and technique of reverse.