逆转录病毒介导DNA修复蛋白在人骨髓细胞中的表达和抗性研究

Expression and drug resistance of human MGMT gene in hemopoietic cells mediated by bicistronic retroviral vector

目的 增强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及耐药基因的特性和在造血细胞保护性基因治疗中的作用和意义。方法 应用RT PCR从人肝组织中获得编码六氧甲基鸟嘌呤 DNA 甲基转移酶 (MGMT)cDNA ,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BCNU加压筛选后的阳性克隆上清经乒乓效应后继而感染K5 62细胞和人造血细胞。应用PCR ,Southernblot,RT PCR ,Westernblot及MTT法检测人MGMT基因在细胞中的转移和表达。结果 酶切鉴定及DNA测序证实其MGMTcDNA克隆的正确性 ,脂质体介导方法成功将其导入病毒包装细胞 ,BCNU加压筛选和乒乓感染法使病毒效价达 8 6× 10 6 CFU ml,逆转录病毒载体介导的MGMT基因在K5 62细胞及人造血细胞中获得有效转移和表达。结论 MGMT耐药基因的成功克隆并导入骨髓造血细胞且获高效表达对开展肿瘤基因治疗的临床研究奠定了实验基础。

Objective To increase myeloid progenitors resistance to chemotherapy and prevent myelosuppression caused by alkylating agents. Methods Total cellular RNA was extracted from human liver and cDNA was synthesized by superscript reverse transcriptase,a polymerase chain reaction(PCR) was conducted. We obtained a full length cDNA fragment encoding human alkyguarine DNA alhyltransferase(MGMT). The PCR product was cloned into pGEMT T vector and further subcloned into G1Na retrovirus expression vector. Then the recombinant plasmid was transduced into the packaging cell lines GP+E86 and PA317 by lipofect AMINE.Results By using the medium containing BCNU for cloning selection and ping ponging supernatant infection between ecotropic produced clone and amphotropic producer clone,we obtained high titer amphotropic PA317 producer clone with the highest titer up to 8.6×10 6 CFU/ml. Human hematopoietic cells were infected repeatedly with this high titer virus under stimulation of hemopoietic growth factors IL 3,IL 6 and SCF.PCR,RT PCR,Southern blot,Western blot and MTT analyses showed that MGMT gene has integrated into the genomic DNA of human hemopoietic cells and expressed efficiently. Conclusion This study provides a foundation for application of gene therapy to tumor clinical trial.