MDR1基因转染K562细胞株及人骨髓干祖细胞的研究

MDR1 gene transfer in K562 cell line and hematopoietic stem progenitor of human bone marrow

目的 研究应用基因工程技术将外源性MDR1基因转染K5 6 2及人骨髓干祖细胞 ,使其获得多药耐药表型 ,抵抗化疗中出现的骨髓抑制的可行性。方法 采用磷酸钙沉淀法建立产病毒包装细胞 ;上清液法分别感染K5 6 2细胞和人骨髓干祖细胞 ;集落筛选法、流式细胞仪、SP免疫组化法检测基因转染率 ;MTT法、柔红霉素泵出实验检测Pgp的功能 ;流式细胞仪分析细胞周期及SP法检测bcl 2、c myc基因表达 ,了解转染行为对靶细胞增殖、凋亡的影响。数据分析用配对t检验。结果 ①被转染的K5 6 2细胞人骨髓干祖细胞最高转染率分别为 34%、35 %。②被转染细胞具有外源性多药耐药功能。③转染未引起异常凋亡及增殖发生。结论 用MDR1基因转染K5 6 2、骨髓干祖细胞是一种稳定、有效、安全的生物治疗方法 ,这项研究为临床应用MDR1基因转染骨髓干祖细胞 ,回输患者体内 ,保护骨髓免遭化疗损害 ,从而为实施大剂量、冲击式化疗 ,提高治愈率奠定基础 ,对最终临床实施MDR1基因治疗辅助化疗有重要意义。

Objective The objective of the study is to evaluate the stability and function of MDR1 gene expression and to assess the feasibility and safety of using MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy. Methods PA317 cells were transfected with retrovirus vector, pHaMDR1/A which contained full-length human MDR1 cDNA, using calcium phosphate precipitation. Human hematopoietic stem progenitor cells were enriched utilizing density grade centrifuge and immunomagnetical beads. The K562 cells and hematopoietic stem progenitor cells were infected with the virions produced by PA317-HaMDR1/A1. The transfection rate was determined using CFU selection, FACS and SP immunohistochemical methods. The foreign Pgp function was tested using MTT assay, CFU selection and DNR exclusion with FACS analysis. The cellular cycle and the expression of bcl-2 and c-myc gene were assayed using PI via FCM and SP stain so that the influence of transfection on target cells proliferation or apoptosis could be determined. Results An amphotropic virus producer cell line, PA317-HaMDR1/A1, was generated with a virus titer of 3.35 ×10 5 CFU/ml. The highest transfection rate of infected K562 cells is 34%. The rate was up to 84% after colchicine selection and the exogenous Pgp expression lasted for about 4 month. Infected K562 cells acquired exogenous MDR function, which was found to be significant ( P < 0.01 ) in chemotherapy. Transfection did not lead to abnormal apoptosis or proliferation. Conclusion The transfer of mdr1 gene into hematopoietic cells offered a potentially useful and safe mean of chemoprotection.