白血病多药耐药裸鼠荷瘤模型的建立

摘要

目的 建立人白血病荷瘤小鼠多药耐药模型.方法 将人白血病细胞K562接种于裸鼠背部皮下,持续低剂量瘤内注射阿霉素,逐步诱导建立耐阿霉素的白血病荷瘤裸鼠模型.绘制裸鼠移植瘤生长曲线;MTT法检测阿霉素诱导后移植瘤细胞对各化疗药物的敏感性;荧光定量PCR检测MDR1、MRP1、LRP1基因表达水平;免疫组化和流式细胞技术检测P-gp、MRP1、LRP1蛋白表达;流式细胞技术检测移植瘤细胞周期分布和细胞内阿霉素浓度.结果 裸鼠皮下移植瘤的成瘤率为100％;化疗组瘤细胞对ADR、VP16、DNR和VCR的IC50均明显高于对照组,其耐药指数分别为40.78、20.59、21.24、18.81;阿霉素诱导组MDR、MRP1、LRP1基因mRNA表达水平分别是对照组的31.55、3.35、3.16倍;免疫组化和流式细胞术检测结果均显示诱导组P-gp、MRP1、LRP1蛋白表达较对照组明显升高;流式细胞术检测显示诱导耐药组瘤细胞S期比例[(8.0±1.32)％]较对照组[(56.92±1.41)％]明显降低,细胞内阿霉素浓度较对照组明显减少.结论 成功建立白血病多药耐药动物荷瘤模型,为克服肿瘤继发多药耐药的深入研究奠定了基础.%Objective To establish the xenograft leukemia model with stable multidrug resistance (MDR) in nude mice induced by low-dose repeated injection of Adriamycin. Methods The nude mice inoculated by K562 cells were injected with Adriamycin of low dosage. The growth curve of the nude mice was drawn during Adriamycin injection. The effects of Adriamycin on K562 cells were assessed by MTT assay. The expression of P-glycoprotein (P-gp) was determined by flow cytometry. The effect of Adriamycin on P-gp activity was evaluated by measuring intracellular Adriamycin accumulation. The expressions of P-gp, MDR-associated protein (MRP1) and lung resistance-related protein (LRP1) genes were detected by fluorescence quantitative polymerase chain reaction. The expressions of P-gp, MRP1 and LRP1 were measured by immunohistochemistry. Results All nude mice were successfully inoculated and tumors were induced. Compared with that in the control group, IC50 of the cell line to the various chemotherapeutic agents was increased after 8 weeks of Adriamycin injection. The expressions of MDR, MRP1 and LRP1 mRNA of the xenografts were higher than those in the control group. The immunohistochemistry results showed that the expressions of P-gp, MRP1 and LRP1 proteins were higher than those in the control group. The number of cells in synthetic phase was greater than that in the control group. The intracellular concentration of Adriamycin was obviously lower than that in the control group. Conclusion We successfully established the multidrug resistant animal model, which establishes groundwork for research on acquired multidrug resistance in tumor.