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目的:探讨将人双突变的二氢叶酸还原酶基因(DHFR) 和胞苷脱氨基酶基因(CD)同时导入小鼠骨髓细胞中, 观察小鼠对大剂量氨甲喋呤(MTX)和阿糖胞苷(Ara-C) 的耐受性,研究骨髓耐受联合化疗的可行性. 方法:以反转录病毒为载体,将人双突变的二氢叶酸还原酶基因(DHFR)和胞苷脱氨基酶基因(CD)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及受体小鼠骨髓移植后经药物处理后的骨髓细胞耐MTX及Ara-C CFU-GM生成情况;转基因小鼠骨髓细胞提取的DNA,用PCR检测转基因小鼠骨髓细胞耐药基因的表达;观察转基因小鼠经大剂量MTX和Ara—C 化疗后血象、体质量及生存率的变化. 结果:骨髓移植前共培养后供体的骨髓细胞和骨髓移植后受体含有耐药基因(SFG—F/S—CD)的骨髓细胞均有耐药克隆的形成(14%,20%;X2分别为42.55,44.26;P<0.01), 并明显增加了对MTX和Ara-C的耐受;与对照组比较含双耐药基因组动物经大剂量化疗后,生存率明显提高(X2=7.42,P<0.01),血象逐渐恢复正常;转基因小鼠骨髓细胞经PCR检测,显示有F/S—CD基因条带;耐药基因转染后小鼠骨髓对MTX和Ara—C的耐受明显增加. 结论:双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和Aar—C的耐药性.
OBJECTIVE: To investigate the effects of high dose methotrexate (MTX) and cytarabine on the proliferation of mouse bone marrow cells by dihydrofolate reductase gene (DHFR) and cytidine deaminase gene (CD) (Ara-C) tolerance to study the feasibility of bone marrow tolerance combined with chemotherapy.Methods: The retrovirus was used as a carrier, the human double mutant dihydrofolate reductase gene (DHFR) and cytidine deaminase The gene (CD) was transfected into mouse bone marrow stem cells by co-culture, and the MTX and Ara-C CFU-GM production of the treated bone marrow cells and the bone marrow cells of the recipient mice after the bone marrow transplantation were observed. The DNA extracted from mouse bone marrow cells was used to detect the expression of drug resistance gene in bone marrow cells of transgenic mice and the change of blood picture, body weight and survival rate after high dose MTX and Ara-C chemotherapy in transgenic mice were observed.Results: The bone marrow cells from the donor co-cultured donor bone marrow and the bone marrow cells containing the drug-resistant gene (SFG-F / S-CD) after BMT were resistant to clones (14%, 20%; X2, 42.55, 44.26; P <0.01), and significantly increased the tolerance to MTX and Ara-C; compared with the control group After high-dose chemotherapy, the survival rate was significantly increased (X2 = 7.42, P <0.01), and the blood was returned to normal. The bone marrow cells of transgenic mice were detected by PCR and showed F / S-CD gene bands. The resistance to MTX and Ara-C in the bone marrow of mice transfected with drug-resistant gene was significantly increased.Conclusion: Dual resistance genes can enter mouse bone marrow cells and obtain co-expression, and improve the resistance of hematopoietic cells to MTX and Aar-C Medicinal properties.