Namalwa 12/MTX耐药株的建立及其耐药机理的初步研究

Primary Study on Establishment of Namalwa 12/MTX ResistantCell Strain and Its Mechanism

背景与目的:甲氨蝶呤(methotrexate,MTX)治疗恶性肿瘤已有50多年的历史,已发现其在不同类型的白血病中具有不同的耐药机制。根据不同的药物作用机制对白血病患者实施个体化用药,以使用药更规范、更科学,不断提高白血病疗效。但尚未见有关其在恶性淋巴瘤中耐药性的阐述,本研究旨在建立Burkitts淋巴瘤MTX耐药细胞株Namalwa12/MTX,并探讨其耐药机理。方法:经MTX脉冲式作用于野生型Namalwa细胞12次产生Namalwa12/MTX耐药株;用MTT法评判野生株及Namalwa12/MTX细胞株对MTX的药效差异;用RT-PCR检测二氢叶酸还原酶(DHFR)mRNA表达量;用3H-MTX掺入及β-液闪仪检测还原性叶酸载体(RFC)功能;用3H-MTX掺入结合HPLC分离法检测两细胞株形成MTX多聚谷氨酸(MTXPG)的能力。结果:Namalwa12/MTX细胞对MTX耐药性较Namalwa细胞提高了20多倍。此耐药性并非RFC功能不良所致,而与形成MTXPG的量密切相关。Namalwa野生株与耐药株Namalwa12/MTX形成的MTXPG1～6分别为(4453±236)pmol/109个肿瘤细胞及(1583±26)pmol/109个肿瘤细胞,MTXPG4～6分别占总MTXPG1～6的25.4%及5.5%(P<0.05);DHFRmRNA表达逐渐增高,并参与Namalwa12/MTX耐药性的形成。结论:MTXPG合成障碍及DHFRmRNA表达增高导致Namalwa12/MTX细胞株对MTX耐药。

Background & Objective:Methotrexate has been used to treat malignant tumor for 50 years. Different mechanisms of methotrexate resistance has been known in various kinds of leukemias. Because of individualized chemotherapy according to different drug metabolism in different leukemia patients,the treatment became more reasonable and scientific and the treatment effect has been gradually improved. However,there was few report about its drug resistance mechanism in lymphoma. This study was designed to establish Burkitts lymphoma methotrexate resistant cell strain—Namlwa 12/MTX and study its resistant mechanism. Methods: Namlwa 12/MTX resistant cell strain was established by repeated impulsed exposure to methotrexate. The difference of the methotrexate effect on Namlwa 12/MTX and Namlwa cell strains were evaluated with MTT method. The level of dihydrofolate reductase (DHFR)mRNA expression was assayed with RT PCR. The function of the reduced folate carrier (RFC) were tested with 3H MTX labeling and counted with β liquid scintillation counter. The capacity to form the polyglutamate methotrexate (MTXPG) in these cell strains was assayed with 3H MTX labeling and high pressure liquid chromatography (HPLC) separation. Results:More than 20 times methotrexate resistance was found in Namalwa 12/ MTX cell strain in comparison with the Namalwa cell strain. This resistance was not associated with the dysfunction of the RFC, but was closely associated with the amount of MTXPG formed. The amount of total MTXPG (MTXPG1 6) formed in these 2 cell strains was (1583±26) pmol/109 tumor cells and (4453±236) pmol/109 tumor cells, respectively (P< 0.05), while the amount of long chain MTXPG (MTXPG4 6) formed only accounted for 5.5% and 25.4% of the MTXPG1 6,respectively (P< 0.05).The level of DHFR mRNA expression was gradually increased with the formation of the drug resistance in Namalwa 12/MTX. Conclusion: Synthesizing difficulty of MTXPG and over expression of DHFR mRNA level result in methotrexate resistance in Namalwa 12/MTX cell strain.