【作者】 刘金超；

【导师】 李书忠；

【作者基本信息】 青岛大学 ， 外科学， 2008， 硕士

【摘要】 目的:建立耐甲氨蝶呤(methotrexate,MTX)的人骨肉瘤U-2 OS细胞亚系,探究还原性叶酸载体(reduced folate carrier,RFC)、二氢叶酸还原酶(dihydrofolatereductase,DHFR)基因表达与MTX耐药的关系。方法:采用大剂量冲击与逐步增加剂量相结合的方法,对人骨肉瘤细胞株U-2 OS进行诱导培养,建立4株耐药细胞株U-2 OS/R1～U-2 OS/R4。MTT法测定各细胞亚系对MTX的敏感性;半定量RT-PCR方法检测各亚系还原性叶酸载体(RFC)、二氢叶酸还原酶(DHFR)mRNA的表达。结果:本实验成功建立了4株MTX耐药细胞亚系U-2 OS/R1～U-2 OS/R4,MTT显示各亚系的耐药性逐渐增加,与其亲代细胞相比耐药指数RF分别为4、23、71、142倍。随体外U-2 OS/R1～4细胞耐药性增加,RFC基因表达逐渐减少,U-2 OS/R1～R4细胞分别为原代U-2 OS细胞的72%、66%、64%、58%(P均＜0.05);DHFR mRNA表达逐渐增强,DHFR mRNA表达水平在U-2 OS/R1～4细胞分别为原代U-2 OS细胞的3.5、6.2、18.0、31.2倍(p均＜0.05)。结论:还原性叶酸载体(RFC)、二氢叶酸还原酶(DHFR)mRNA共同参与了U-2 OS骨肉瘤细胞MTX耐药,MTX耐药与RFC基因mRNA表达降低及DHFR基因mRNA表达增加相关。这种现象可能存在于临床的骨肉瘤病人,因而在诊断时测定RFC、DHFR基因状态将有助于筛选MTX耐药病人;而且这些耐药细胞亚系为进一步研究骨肉瘤MTX耐药及逆转提供了一种新的模型。更多还原

【Abstract】 Objective:To evaluate the impact of reduced folate carrier(RFC) and dihydrofolate reductase(DHFR) genes on methotrexate(MTX) resistance in osteosarcoma cells(U-20S).Methods:Four MTX-resistant variants(U-2 OS/R1～U-2 OS/R4) were obtained by exposing parental cell line to in vitro high dose and stepwise increased MTX concentrations.The sensitivity of the MTX resistance was measured by MTT assay;the level of reduced folate carrier(RFC) and dihydrofolate reductase(DHFR) mRNA expression was assayed with RT-PCR.Results:The authors developed four MTX-resistant variants(U-2 OS/R1～U-2 OS/R4) that were resistant to MTX,the resistance factor(RF) in U-2 OS/R1～U-2 OS/R4 is 4,23,71,142 competed with the U-2 OS cell.RFC gene mRNA expression level of resistant cells was decreased gradually along with the concentration increasing of MTX in U-2 OS/R1～U-2 OS/R4 cell lines(72%、66%、64%、58%)(p＜0.05);with DHFR gene mRNA expression level increased 3.5,6.2, 18.0,31.2 folds(p＜0.05).Conclusions:The MTX resistance was associated with increased levels of DHFR and decrease of RFC gene expression.The DHFR and RFC gene expression level result in MTX resistance in human osteosarcoma cell U-2 OS which can be present at clinical onset in osteosarcoma patients.Simultaneous evaluation of RFC, DHFR gene status at the time of diagnosis may help for the identification of potentially MTX-unresponsive osteosarcoma patients;moreover these MTX resistent osteosarcoma cell sublines are useful models for further research of MTX resistence in osteosarcoma and for the development of treatment protocols.更多还原