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目的:多药耐药基因(MDR1)的异常扩增和过度表达是肿瘤细胞产生多药抗药性的重要原因之一。本研究试图将MDR1导入骨髓或外周血造知干细胞并使入获得耐药表型,以用于减轻大剂量化疗对造血细胞的毒性。方法:应用反转录病毒载体pHaMDR1/A将MDR1基因导入人外周血造血干细胞。结果:用流式细胞法没得导入率为3.74％。PCR法检测转染后造血干细胞中外源MDR1阳性。PCR法检测转染细胞所形成的CFU—GM集落中,外源MDR1阳性比例为1/4;而未转染细胞所形成的CFU—GM集落均为阴性。集落形成试验证明,转染和非转染细胞的克隆形成能力相似,说明基因转染不影响血干细胞的增殖能力,但转基因造血干细胞对人疗药物紫杉醇耐受性增加。结论:上述结果表明应用逆转录病毒载体将MDR1基因导入人外周血造血干细胞是可行的。
OBJECTIVE: Abnormal amplification and overexpression of multidrug resistance gene (MDR1) is one of the important reasons for the multidrug resistance of tumor cells. In this study, we attempted to introduce MDR1 into bone marrow or peripheral blood to make stem cells and obtain resistant phenotype to reduce the hematopoietic toxicity of high-dose chemotherapy. Methods: MDR1 gene was introduced into human peripheral blood hematopoietic stem cells using retroviral vector pHaMDR1 / A. Results: The flow cytometry method was not introduced rate of 3.74%. PCR detection of exogenous MDR1 positive in hematopoietic stem cells after transfection. The CFU-GM colonies formed by transfected cells were detected by PCR. The positive rate of exogenous MDR1 was 1/4, while the CFU-GM colonies formed by untransfected cells were all negative. Colony formation assay showed that the clonogenic capacity of transfected and nontransfected cells was similar, indicating that gene transfection did not affect the proliferation of blood stem cells, but the transgene hematopoietic stem cells increased paclitaxel tolerance to human therapy. Conclusion: The above results indicate that it is feasible to introduce MDR1 gene into human peripheral blood stem cells by retroviral vector.