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双突变二氢叶酸还原酶基因对小鼠骨髓细胞化疗保护作用的体外研究

Research of chemoprotection through transferring double-mutant dihydrofolate reductase gene into mice bone marrow cells in vitro
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摘要 目的探讨将人双突变的二氢叶酸还原酶基因(dihydrofolate reductase,DHFR)导入小鼠骨髓细胞中,观察小鼠骨髓细胞对大剂量甲氨蝶呤(methotrexate,MTX)的耐受性,研究骨髓耐受大剂量化疗的可行性。方法以反转录病毒为载体,将DHFR通过共培养转染入小鼠骨髓干细胞,观察共培养后的骨髓细胞耐MTX CFU-GM生成情况;小鼠骨髓细胞提取的DNA,用PCR检测小鼠骨髓细胞耐药基因的表达。结果含有耐药基因(SFG-F/S)的骨髓细胞耐药克隆为16%,对照组为0,χ2=47.88,P<0.005;转基因小鼠骨髓细胞经PCR检测,显示有F/S基因条带;耐药基因转染后小鼠骨髓细胞对MTX的耐受明显增加。结论耐药基因可以进入小鼠骨髓细胞并且获得共表达,提高了造血细胞对MTX的耐药性。 OBJECTIVE: To explore the feasibility of transferring gene of dihydrofolate reductase (DHFR) into mouse bone marrow cells, to observe the drug resistance of high dose MTX and to improve the tolerance of myelosuppression following chemotherapy. METHODS: Human double-mutant dihydrofolate reductase gene was transferred into mouse bone marrow cells by retroviral vector. Resistant colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in mouse bone marrow cells by retroviral infection after treatment by drugs. DNA was extracted from mouse bone marrow cells. The expression of drug resistant genes in mouse bone marrow cells after transferring by retroviral vector was checked by PCR. RESULTS: Bone marrow cells after cocultured with the retroviral producer cells transduced with the genes (SFG-F/S) showed the drug resistance colonies (16%) to MTX, χ2=47.88, P<0.005. Expression of DHFR gene in extracted DNA of transfected mice marrow cells were demonstrated by PCR. CONCLUSION: DHFR drug resistance gene can not only integrate and co-express in mice bone marrow cells but also increase the drug resistance to MTX.
出处 《肿瘤防治杂志》 2005年第14期1066-1068,共3页 China Journal of Cancer Prevention and Treatment
关键词 四氢叶酸脱氢酶 甲氨蝶呤 鼠科 骨髓 体外研究 tetrahydrofolate dehydrogenase methotrexate bone marrow in vitro
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参考文献12

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