多药耐药基因转染骨髓单个核细胞移植联合化疗治疗乳腺癌的实验研究

Transplantation of hMDR1-transferred bone marrow mononuclear cells and chemotherapy in breast cancer xenograft in mice

目的研究外源性人多药耐药基因(human multidrug resistance gene 1,hMDR1)转染骨髓单个核细胞(mono-nuclear cells,MNCs)移植联合超剂量表阿霉素化疗在4T1乳腺癌治疗中的骨髓保护和治疗作用。方法用携带目的基因hMDR1和绿色荧光蛋白(green fluorescence protein,GFP)报告基因的复制缺陷型重组腺病毒(rAd-hMDR1-GFP)转染原代培养的MNCs以获取hMDR1基因修饰的MNCs(MNCs-rAd-hMDR1-GFP),将修饰后的MNCs移植到4T1乳腺癌模型体内,在表阿霉素超剂量化疗中观察骨髓造血功能的保护和肿瘤的治疗效果。结果 hMDR1基因成功转入小鼠骨髓单个核细胞,体外转染率达26.26%;转染细胞移植后能定植于受体骨髓中并功能性表达。在超剂量化疗中,hMDR1基因能有效保护受体骨髓的造血功能,荷瘤动物能耐受3倍剂量表阿霉素化疗,外周血白细胞数能维持在(5.7±0.6)×109/L;同时超剂量化疗能迅速杀灭肿瘤细胞,使荷瘤动物存活时间明显延长(P<0.05),治愈率明显提高(P<0.05)。结论 hMDR1基因转染骨髓造血细胞移植能明显提高受体骨髓对化疗药物的耐受性,联合超剂量化疗能快速、有效的杀灭肿瘤细胞,提高恶性肿瘤的治愈率。

Objective To determine the myeloprotection and therapeutic effects of transplantation of human multidrug resistance gene 1 （hMDR1）-transferred mononuclear cells （MNCs） on over-dosage chemo- therapy to the mice with 4T1 xenograft breast cancer. Methods Primary MNCs isolated from bone marrow of BALB/c mice were transfected with a replication-deficient recombinant adenovirus expression vector carrying hMDR1 and GFP （rAd-hMDR1-GFP） to obtain hMDRl-modified MNCs （MNCs-rAd-hMDR1-GFP）. The transfected and nontransfected cells were then transplanted into BALB/c mice with 4T1 breast cancer xenograft and over-dose epirubicin （15 mg/kg, once a week）. The xenograft mice without any treatment （control）, with normal dose epirubicin （5 mg/kg, once a week） and with over-dose epirubicin served as control. The chemoprotection effects of hMDR1 gene for the hematopoietic bone marrow and effectiveness of over-dose chemotherapy were assessed. Results MNCs were transfected with the vector rAd-hMDR1-GFP in vitro with a transfection rate of 26.26%. After transplantation, the expression of hMDR1 gene, which confers chemoprotection, was confirmed. The mouse with 4T1 breast cancer xenograft could tolerate the 3 times higher than the usual dose of epirubicin and the leukocytes count in peripheral blood was maintained at （5.7 ±0.6）×10^9/L. Over-dose chemotherapy reduced the breast cancer cells dramatically and extended the survival time of mouse （P 〈 0. 05）. Conclusion Transplantation of hMDR1 transfected bone marrow hematopoietic cells increases the tolerance of xenograft to chermotherapy and improves the therapeutic effect of chemotherapy to the tumor.