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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cDNAmdr1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(HL60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MNC2×106/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mdr1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HL60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HL60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?
Objective To investigate the effect of mdr1 gene-transfected cord blood mononuclear cells (MNCs) on the myeloprotection in acute myeloid leukemia mice. Methods The mdr1 gene containing human full-length cDNA was introduced into cord blood MNC by retrovirus-mediated method, that is, the retrovirus supernatant was co-cultured with cord blood MNC in vitro. The SCID of human myeloid leukemia cell line (HL60) Mice were divided into 3 groups A (observation group), mice were injected intravenously with mdr1 cord blood MNC2 × 106 / mouse, a total of 2 times; B, C two control mice at the same dose, respectively, were injected untransfected mdr1 cord blood MNC and equal volume of saline. Three groups of leukemia SCID mice were treated with increasing doses of harringtonine every week. The number of leukocytes in peripheral blood, the positive rate of tumor cells, the histopathology and the surface antigens of HL60 cells (CD33) Tolerance of anti-cancer drugs and anti-tumor effect in mice. At the same time, the expression and function of mdr1 gene in mice were detected by PCR, immunohistochemistry and daunorubicin excretion respectively. Results ① In vitro, the mdr1 gene was successfully transfected into cord blood MNC and the transfection rate was about 30%. ② The inoculation of 2 × 106 HL60 cells into the sublethally irradiated SCID mice resulted in successful leukemia animal model. Transplantation of transgenic mice with mdr1 gene successfully established murine model of mdr1 transgenic cord blood cell transplantation, and can be used as a pre-clinical evaluation of leukemia mdr1 gene to protect the bone marrow; ④ transgenic cord blood cell transplanted mouse homoharringtonine tolerance higher than normal 5 to 6 times the dose of peripheral blood leukocytes maintained at about 3.0 × 109 / L, peripheral blood smear tumor cells down to 5%?