多药耐药基因转染的脐血细胞对白血病小鼠化疗的骨髓保护作用

Protection of mdr1 transfected cord blood mononuclear cell graft against anticancer agents in vivo

目的探讨转染多药耐药(mdr1)基因的脐血单个核细胞(MNC)对急性髓系白血病小鼠的骨髓保护作用及疗效.方法通过逆转录病毒介导的方法将含有人全长cDNA mdr1基因导入脐血MNC,即逆转录病毒上清液与脐血MNC体外共培养;将接种人髓系白血病细胞系(HL-60细胞)的SCID小鼠分成3组:A组(观察组)经鼠尾静脉注射转染mdr1的脐血MNC 2×106/只,共2次;B、C两对照组小鼠以同样剂量、方法分别注射未转染mdr 1的脐血MNC和等容积的生理盐水.3组白血病SCID小鼠在每周递增高三尖杉酯碱剂量化疗下,通过检测小鼠外周血白细胞数、瘤细胞阳性率、组织病理和HL-60细胞表面抗原(CD33)等观察转基因小鼠和对照小鼠对抗癌药物的耐受性及抗肿瘤疗效.同时分别采用PCR技术、免疫组化方法和柔红霉素排出试验检测mdr1基因在小鼠体内的表达和功能.结果①体外成功地将mdr1基因导入脐血 MNC,转染率达30%左右;②用HL-60细胞2×106接种于经亚致死量照射的SCID小鼠可成功制成白血病动物模型;③采用程序性移植转基因细胞方法成功建立了mdr1转基因脐血细胞移植小鼠模型,并可作为白血病临床前期体内评价mdr1基因保护骨髓作用;④转基因脐血细胞移植小鼠对高三尖杉酯碱耐受性高于正常剂量的5～6倍,外周血白细胞维持在3.0×109/L左右,外周血涂片瘤细胞降至5%以下,病理切片主要脏器未见瘤细胞,CD33+细胞率低于5%,证明进行转基因脐血细胞移植的白血病小鼠在化疗中有明显的骨髓保护作用;⑤PCR技术、免疫组化方法、柔红霉素排出试验证实mdr1基因在体内可有效整合及表达.结论基因转染脐血MNC移植到荷瘤鼠体内可在化疗中保护骨髓细胞免受抗癌药物的损伤.

Objective To explore the myeloprotection effect of mdr1 transfected cord blood cells (CBMNCs) graft against high-dose homoharringtonine leukemia-bearing severe combined immunodeficient (SCID) mice model.Methods Multidrug resistant(mdr1)gene was transfered into CBMNCs by a retrovirus vector, containing full-length cDNA of human mdr1 gene. CBMNCs and high-titer retrovirus supernatant were cocultured with cytokine combinations for 5~6 days. The SCID mouse models bearing human HL-60 cell leukemia were divided into three groups. Group A received tail vein injection of 2×10~6 mdr1 gene transduced CBMNCs at day 1 and 3, groups B and C 2×10~6 untransduced CBMNCs and same volume of normal saline, respectively. The 3 groups of the mouse model were treated with weekly escalated doses of homoharringtonine. The peripheral white blood cell(WBC) counts, the human leukemia cells percentage in peripheral blood, the histological findings of main organs were assayed. The CD33 positive HL-60 cells in bone marrow were determined by flow cytometry. The function and expression of mdr1 gene were examined by PCR, immunochemistry(IC) and DNR extrusion test in vivo.Results ①mdr1 gene was transferred into CBMNCs successfully and the transfection frequency was 30%. ② Leukemia SCID mice were xenotransplanted with mdr1-transfected BMMNCs by a programmed procedure and could be used as a valuable model for in vivo evaluating myeloprotection effects. ③ The transfected mice could tolerate homoharringtonine 5~6 folds higher than conventional dose and kept peripheral WBC count at a mean of 3×10~9 /L,with the peripheral human myeloid leukemia cells percentage decreasing to less than 5%. Histological examination showed that there was no leukemia infiltration in the main organs, the CD33 positive HL-60 cells in bone marrow were less than 5%. ④The repopulation frequency of the transfected CBMNs in marrow were 9.13%. DNR extrusion test confirmed that the P-gp product maintained its biological function in the marrow.Conclusion mdr1 transferred-human CBMNC can xenotransplanted and repopulated in leukemia-bearing SCID mouse and are protected from chemotherapy-induced myelosuppression.