多药耐药基因转染脐血单个核细胞及其表达与功能的体外研究

Multidrug resistant gene transfer into human cord blood mononuclear cells in vitro and its expression and function

目的 为克服白血病化疗中的骨髓抑制 ,采用含人多药耐药 (MDR1)基因全长cDNA逆转录病毒载体转染人脐血单个核细胞 ,探索一种MDR1基因导入脐血造血细胞稳定、有效的方法 ,评价MDR1基因转染脐血细胞及其功能的表达 ,为体内转染MDR1基因保护骨髓免受大剂量化疗药物损伤提供条件。方法 采用含有MDR1基因表达质粒pHaMDR1/A的包装细胞PA3 17传代培养产病毒法 ,通过浓缩病毒上清液将MDR1基因导入人脐血单个核细胞 ,应用聚合酶链反应 (PCR)方法、免疫组化法、流式细胞学等方法从基因、蛋白质、功能及细胞生物学特性等不同水平检测MDR1基因在脐血单个核细胞的表达、转染率及其与转染时间的关系和功能的表达 ;转染行为是否影响脐血细胞生物学特性。结果 (1)建立了一种安全可行、稳定高效的MDR1基因体外转染脐血造血细胞的体系和方法 ;(2 )PCR方法证实外源性MDR1基因可有效地在体外整合到脐血单个核细胞中 ;(3 )免疫组化法测得 2日、4日、6日转染率分别为 18 0 %、2 9 8%、3 4 7% ;(4 )柔红霉素排出试验证实导入的MDR1基因表达产物P gp有正常的生物学功能 ;(5 )转染后的脐血细胞周期生物学特性无异常。结论 MDR1基因体外转染脐血造血细胞是安全可行的 ,且在体外有稳定、有效的表达。这一方法为进一步在化疗骨?

Objective The investigators aimed to transfer multidrug resistant (MDR1) gene into hematopoietic cells of human cord blood mononuclear cells (CBMNC) by a retrovirus vector containing a full-length cDNA of human MDR1 gene and explored a secure, stable and efficient method. The authors also attempted to assess the safety, feasibility and functional expression of transferred MDR1 gene into cord blood mononuclear cells in vitro and provided with favorable conditions for protecting bone marrow hematopoietic cells from damage of the drug in vivo undergoing high-dose anticancer agent. Methods MDR1 gene was transferred into mononuclear cells of human cord blood mononuclear cells with plasmid pHaMDR1/A by the retrovirus-mediated vector. The expression of MDR1 gene in the cord blood mononuclear cells in vitro, the positive transferring rate on different transferring terms and the P-gp function were tested by PCR, immunohistochemistry (IC) and flow cytometry (DNR extrusion test) at the different levels of gene, protein and molecule, respectively. The cell cycle of the CBMNC after transduction was tested by flow cytometry. Results (1) A safe, stable and efficient method of transferring MDR1 gene into human CBMNC was established successfully. (2)The effective integration of MDR1 gene in human CBMNC could be tested by PCR method. (3)After 2 days, 4 days and 6 days of transduction the positive transferring rates of CBMNC were 18.0%, 29.8% and 34.7%, respectively. (4) It was confirmed by DNR extrusion test that the product P-gp of transferred MDR1 gene maintained its biological function. (5) The cell cycle of transferred CBMNC conserved normal biological characteristics. Conclusion MDR1 gene transfer into human CBMNC was safe and available. The stable and efficient expression of MDR1 gene could be tested in vitro. These data provided a foundation and evidence for the experiment on preventing bone marrow from toxicity of anticancer drug.