DNA修复蛋白MGMT基因的克隆及转导脐血CD23^＋细胞后耐药特征的研究

目的：增强脐血ＣＤ３４＾＋造血细胞对化疗药物的耐药表型，探讨逆转录病毒介导的基因转移效率和耐药基因特性，以及在脐血造血干细胞保护性基因治疗中的作用和意义。方法：应用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）从人肝细胞中获得编码六氧甲基鸟嘌呤－ＤＮＡ－甲基转移酶（Ｏ＾６－ｍｅｔｈｙｌｇｕａｎｉｎｅ－ＤＮＡ－ｍｅｔｈｙｌｔｒａｎｓｆｅｒａｓｅ，ＭＧＭＴ）ｃＤＮＡ；利用基因重组技术，将其克隆于ｐＧＥＭ－Ｔ质粒载体并构建了逆转录病毒载体Ｇ１Ｎａ－ＭＧＭＴ；应用脂质体ＬｉｐｏｆｅｃｔＡＭＩＮＥ基因转移法将后者导入ＧＰ＋Ｅ８６和ＰＡ３１７病毒包装细胞，以卡氮芥１，３－Ｂｉｓ（２－Ｃｈｌｏｒｏｅｔｈｙｌ）－１－Ｎｉｔｒｏｓｏｕｒｅａ（ＢＣＮＵ）加压筛选后的阳性克隆上清经乒乓效应后继而感染脐血ＣＤ３４＾＋细胞。应用ＰＣＲ，Ｓｏｕｔｈ

In vitro study on transduction of human O(6)-methylguanine-DNA-methyltransferase cDNA into human umbilical cord blood CD34(+) cells]

OBJECTIVE: To explore human umbilical cord blood hematopoietic progenitor cells transduced with human O(6)-methylguanine-DNA-methyltransferase (MGMT) gene increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea(BCNU). METHODS: The present authors obtained a full length cDNA fragment encoding the human MGMT from a patient with cholelithiasis liver tissue by RT-PCR method and confirmed by DNA sequencing. The fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the G1Na-MGMT was transfected into the packaging cell lines GP+E86 and PA317 by LipofectAMINE method; using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, the authors obtained high titer amphotropic PA317 producer clone with the highest titer up to 1.6x10(6) CFU/ml. Cord blood CD34(+) cell were transfected repeatedly with supernatant of retrovirus containing human MGMT cDNA under stimulation of hemopoietic growth factors. RESULTS: PCR, RT-PCR, Southern blot, Northern blot, Western blot and MTT analyses showed that MGMT gene had been integrated into the genomic DNA of cord blood CD34(+) cells and expressed efficiently in the transfected cells. The transgene recipient cells conferred 4 folds stronger resistance to BCNU than that of the non-transduced. CONCLUSION: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34(+) cells and expression could confer the resistance of transgene cells to BCNU toxicity.