多药耐药mdr-1基因体外转染兔骨髓造血细胞及其表达与功能的研究

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(daunorubicin DNR)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-g;ucprpteom P-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22％、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基因导人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。

Study on the expression and function of the mdr-1 gene transferred into hematopoietic cells of rabbit bone marrow in vitro

Objective:To explore the optimal transfection conditions in which the concentrated virus supernatant transfer the foreign mdr-1 gene into hematopoietic cells of rabbit bone marrow,and test the expression and function of the mdr-1 gene in cells.Methods:An amphotropic virus producer cell line,PA317-HaMDR1/A1 which contains a full-length cDNA of human mdr-1 gene,was screened with colchicine(90ng/ml),and the supernatant was collected and concentrated.The bone marrow cells of the New Zealand rabbits were gathered and the mononuclear cells which contain many hematopoietic cells were enriched.The hematopoietic cells were cocultivated with concentrated virus supernatant and interleukins.The transfection rate was determined by SP immunohistochemical methods;the integration of mdr-1 gene in rabbit mononuclear cells was tested by PCR method ,and the physiological function of mdr-1 gene was tested by DNR extrusion test.Results:After screening by colchicine,the expression of P-glycoprotein(P-gp) of PA317-HaMDR1/A1 was enhanced.The foreign mdr-1 gene was transferred into hematopoietic cells of rabbit bone marrow successfully,and the transduction rate of 2 days,4days and 6 days were 22%,37%,and 39% respectively.However,the state of the 4 days was the best.The transferred mdr-1 gene had its physiological function.Conclusion:The foreign mdr-1 gene can be transferred into hematopoietic cells of rabbit bone marrow successfully by concentrated virus supernatant transfection method,and the transferring gene can be expressed stably and effectively.The study has provided a basis for the further research on chemoprotection experiment of the mdr-1 gene transferred into the bone marrow cells.