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目的探讨同时导入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)小鼠骨髓细胞对大剂量甲氨蝶呤(MTX)和阿糖胞苷(Ara-C)的耐受性及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DHFR和CD通过共培养转染入两只小鼠骨髓干细胞,观察共培养后的骨髓细胞经Ara-C、MTX、Ara-C+MTX处理后,耐MTX及Ara-C粒-巨噬细胞集落形成单位(CFU-GM)生成情况;转基因小鼠骨髓细胞提取的DNA,用聚合酶链反应(PCR)检测转基因小鼠骨髓细胞耐药基因的表达。结果含有耐药基因(SFG-F/S-CD)的骨髓细胞均有耐药克隆的形成,耐Ara-C、MTX、Ara-C+MTX克隆分别为56%、22%和14%,并明显增加了对MTX和Ara-C的耐受(P<0.005);转基因小鼠骨髓细胞经PCR检测,显示有F/S、CD基因表达;耐药基因转染后小鼠骨髓细胞对MTX和Ara-C的耐受明显增加。结论双耐药基因可以导入小鼠骨髓细胞并且获得表达,提高了造血细胞对MTX和Aar-C的耐药性。
OBJECTIVE: To investigate the effects of DHFR and cytidine deaminase (CD) mouse bone marrow cells on high-dose methotrexate (MTX) and cytarabine (Ara-C ) Tolerance and the feasibility of combined chemotherapy of bone marrow tolerance. Methods Recombinant human double mutant DHFR and CD were transfected into two mouse bone marrow stem cells by co-culture using retrovirus as a vector. The co-cultured bone marrow cells were treated with Ara-C, MTX and Ara-C + MTX (MTT) and Ara-C granulocyte-macrophage colony forming unit (CFU-GM). The DNA extracted from the transgenic mouse bone marrow cells was used to detect the resistance gene of transgenic mouse bone marrow cells by polymerase chain reaction expression. Results All the myeloid cells with drug resistance gene (SFG-F / S-CD) were resistant to clones. The resistance to Ara-C, MTX and Ara-C + MTX was 56%, 22% and 14% Significantly increased the tolerance to MTX and Ara-C (P <0.005). The bone marrow cells of transgenic mice were detected by PCR and showed F / S and CD gene expression. Ara-C tolerance increased significantly. Conclusion Double resistance gene can be introduced into mouse bone marrow cells and expressed, and improve the resistance of hematopoietic cells to MTX and Aar-C.