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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 enhance the resistance phenotypes of hematopoietic cells to chemotherapeutic agents and investigate the efficiency of retrovirus-mediated gene transfer and the characteristics of drug resistance genes and their role and significance in hematopoietic protection gene therapy. Methods The cDNA coding for hexamethyloguanine DNA methyltransferase (MGMT) was obtained from human liver tissue by RT PCR and cloned into the pGEM T plasmid vector and the retroviral vector G1Na MGMT was constructed. The liposome LipofectAMINE was used. The latter was introduced into GP +E86 and PA317 virus packaging cells by gene transfer method. The positive clone supernatant after BCNU pressurization screening was followed by infection of K5 62 cells and human hematopoietic cells via the ping-pong effect. The human MGMT gene was detected by PCR, Southern blot, RT PCR, Western blot and MTT assay. Results The restriction enzyme digestion and DNA sequencing confirmed the correctness of the MGMT cDNA clone. The liposome-mediated method successfully introduced it into virus packaging cells. The BCNU pressure screening and ping-pong infection method resulted in a virus titer of 8 6×10 6 CFU ml. The retroviral vector-mediated MGMT gene was efficiently transferred and expressed in K562 cells and human hematopoietic cells. Conclusion The successful cloning and introduction of MGMT gene into bone marrow hematopoietic cells and high expression of MGMT have laid a foundation for the clinical research on tumor gene therapy.