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本文用转染了人突变DHFR基因逆转录病毒载体的新一代产病毒细胞AM12-S31,研究了其耐药特性、产生的病毒滴度及DHFR基因在小鼠造血细胞的表达.MTT法显示AM12-S31细胞对G418和氨甲喋呤耐受,对照细胞AM12对G418和MTX敏感.产生的病毒滴度为7.8×10~4～4.2×10~5CFU/ml,且为无复制活性病毒.将AM12-S31上清转染小鼠骨髓造血细胞后进行体内、外研究.CFU-GM分析显示:于不同C418浓度均有阳性克隆,而AM12上清转染组则无耐G418克隆.将转染后骨髓细胞从尾静脉回输给经致死剂量(900 rad)照射小鼠,MTX筛选(第一周为4mg/kg,每周二次;以后各周10mg/kg,每周二次),结果:实验组小鼠白细胞计数和红细胞压积逐渐恢复正常;对照组小鼠 30天内全部死亡.PCR分析耐G418 CFU-GM克隆和实验组小鼠骨髓细胞,均检测到标志基因Neo~R的存在.因此,初步研究表明该产病毒细胞能够产生高滴度、安全有效的非复制型逆转录病毒,并能成功转染小鼠造血细胞、表达目的基因,使在MTX筛选条件下重建小鼠造血功能.该研究为进一步开展耐药基因治疗打下了基础.
In this study, a new generation of virus-producing cells, AM12-S31, transfected with a human DHFR gene retroviral vector, was used to study its drug resistance, virus titer and DHFR gene expression in mouse hematopoietic cells. MTT assay showed AM12 -S31 cells were tolerant to G418 and methotrexate, and control cells AM12 were sensitive to G418 and MTX. The virus titer produced was 7.8×10~4~4.2×10~5 CFU/ml, and it was a replication-free virus. AM12-S31 The supernatant was transfected into bone marrow hematopoietic cells in vitro and in vivo. CFU-GM analysis showed positive clones at different concentrations of C418, whereas the AM12 supernatant transfection group had no G418-resistant clones. The bone marrow cells were transfected. The mice were irradiated back to the lethal dose (900 rad) from the tail vein and screened with MTX (4 mg/kg for the first week, twice weekly; 10 mg/kg for each subsequent week, twice per week). Results: Experimental mice The white blood cell count and hematocrit gradually returned to normal; mice in the control group died within 30 days. PCR analysis of the resistant G418 CFU-GM clone and the experimental group of mouse bone marrow cells detected the presence of the marker gene Neo~R. Therefore, a preliminary study was conducted. Shows that the virus-producing cells can produce high titer, safe and effective non-replicating reversal Viruses, and successfully transfected into mouse hematopoietic cells, expression of the target gene, the mouse hematopoietic function reconstruction in the MTX filter criteria. This study further develop drug resistance gene therapy foundation.