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目的 :探讨浓缩病毒上清转染法在体外将多药耐药mdr - 1基因转入兔骨髓造血细胞的条件及检测转染后mdr - 1基因在兔骨髓造血细胞中的表达及功能。方法 :用秋水仙碱 (90ng/ml)筛选含人类全长mdr - 1cDNA的产病毒包装细胞PA317-HaMDR1/A1后进行细胞培养并制备浓缩病毒上清。采集新西兰大白兔骨髓并分离富含造血细胞的单个核细胞。骨髓造血细胞与浓缩病毒上清及细胞因子组合共培养。采用免疫组织化学法检测转染率 ,PCR法检测外源性mdr- 1基因的整合 ,柔红霉素 (daunorubicinDNR)泵出试验检测导入的mdr - 1基因的功能。结果 :秋水仙碱筛选后 ,产病毒包装细胞P -糖蛋白 (P - glycoproteinP - gp)表达增强 :外源性mdr - 1基因能成功的导入兔骨髓造血细胞 ,转染 2日、4日、6日的转染率分别为 2 2 %、37%、39% ,但转染 4日组细胞生长状态最好 ;转入的mdr - 1基因能发挥药物外排泵的功能。结论 :采用浓缩病毒上清转染法能成功的将外源性mdr - 1基因导入兔骨髓造血细胞中并获得稳定的功能性表达 ,为进一步研究mdr- 1基因转染骨髓造血细胞后自体回输在大剂量化疗中对骨髓保护作用的研究提供了依据。
OBJECTIVE: To investigate the conditions of transfection of multidrug resistance mdr - 1 gene into rabbit bone marrow hematopoietic cells in vitro and the expression and function of mdr - 1 gene in rabbit bone marrow hematopoietic cells after transfection. Methods: PA317-HaMDR1 / A1 virus-producing packaging cells containing full-length mdr-1 cDNA of human were screened with colchicine (90ng / ml), and then the cell culture was performed and the concentrated virus supernatant was prepared. New Zealand white rabbits bone marrow harvest and separation of hematopoietic cells rich in mononuclear cells. Bone marrow hematopoietic cells were co-cultured with concentrated virus supernatant and cytokines. The transfection efficiency was detected by immunohistochemistry. The integration of exogenous mdr - 1 gene was detected by PCR and the function of mdr - 1 gene was detected by daunorubicin DNR pumping test. Results: After colchicine screening, the expression of P - glycoprotein P - gp was enhanced: the exogenous mdr - 1 gene could be successfully transfected into rabbit bone marrow hematopoietic cells and transfected on the 2nd, 4th, The transfection rates on the 6th day were 22%, 37% and 39%, respectively. However, the cell growth was the best on the 4th day after transfection. The transfected mdr - 1 gene could exert the function of drug efflux pump. CONCLUSION: The exogenous mdr - 1 gene can be successfully transfected into rabbit bone marrow hematopoietic cells by using concentrated virus supernatant transfection method, and stable functional expression can be achieved. To further investigate the effect of mdr - 1 gene transfection on bone marrow hematopoietic cells In the high-dose chemotherapy in bone marrow protection provides a basis for the study.