提高mdr1基因体外转染骨髓造血细胞效率的实验研究

Research on improving the efficiency of transferring the mdrl geneinto bone marrow hematopoietic cells of rabbit in vitro

目的:探讨mdr1基因体外转染兔骨髓造血细胞的条件及转染后在细胞中的功能性表达。方法:秋水仙碱(90ng/ml)筛选含人mdr1cDNA的产病毒包装细胞PA317-HaMDR1/A,制备浓缩病毒上清液并转染兔骨髓单个核细胞;分别用PCR法、免疫组织化学法和柔红霉素(daunorubicin DNR)排出试验检测mdr1基因的整合及功能性表达。结果:经秋水仙碱筛选后,产病毒包装糖蛋白(P-glycoprotein P-gp)表达增强;外源性mdr1基因能成功的导入兔骨髓造血细胞,转染2日、4日的转染率分别为22%、37%;转入的mdr1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mdr1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mdr1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 explore the optimal transfection conditions in which the-foreign mdrl gene is transferred into hematopoietic cells of rabbit bone marrow, and test the expression and function of the mdrl gene in the ceils. Methods： An amphotropic virus producer cell line, PA317 - HaMDR l/A, which contains a full- length cDNA of human mdrl gene, was screened with colehicines（90ng/ml）,and the supernatant was collected and concentrated to cocultivated with the bone marrow mononuclear cells of the rabbit. The integratian, transfection rate and physiological function of mdr 1 gene was tested by PCR, SP immunohistochemical method and DNR extrusion test respectively. Results：After screening by colchicinesj the expression of P- gly, coprotein （p- gp）of PA317 - HaMDR1/A was enhanced. The foreign mdr 1 gene was transferred into hematopoietic ceils of rabbit bone marrow successfully, and the transduction rate of 2 days and 4 days were 22 %, 37 %. The transferred mdr 1 gene had it＇ s physiological function. Conclusion ： The foreign mdr 1 gene can be transferred into hematopoietic cells of rabbit bone marrow successfully by concentrated virus supematant transfection method, and the transferred gene can be expressed stably and effectively. The study has provided a basis for the further research on chemopmtection experiment of the mdr 1 gene transferred into the bone marrow cells.