重组人GM-CSF逆转录病毒基因转移系统的建立及其在人肿瘤细胞中的稳定表达  

Recombinant human GM-CSF retroviral vector construction and expression in human breast cancer cells

作　　者：赵明[1] 毛宁[1] 巴德年[2] 张明伟[1] 杜德林[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]中国医学科学院中国协和医科大学

出　　处：《中华肿瘤杂志》1997年第1期42-44,共3页Chinese Journal of Oncology

摘　　要：目的　建立逆转录病毒介导的 GM- CSF基因转移系统 ,为 GM- CSF基因修饰的肿瘤细胞疫苗研究奠定实验基础。方法　应用基因重组技术将 GM- CSF c DNA克隆于逆转录病毒载体p DORneo,获得重组载体 p DORGM。经脂质体介导将其转染于病毒包装细胞系 PA317细胞 ,经NIH3T3细胞检测病毒滴度 ,NIH3T3放大实验检测辅助病毒 ,并用高滴度病毒感染人乳腺癌细胞系MCF- 7。结果　GM- CSF基因克隆入逆转录病毒载体 ,经 PA317细胞包装产生病毒 ,最高的病毒滴度为 1× 10 6CFU/ ml,且没有发现辅助病毒存在。重组病毒感染的人乳腺癌 MCF- 7细胞在体外长期培养中保持稳定分泌 GM- CSF,活性在 50 0～ 80 0 U/ 10 6细胞 / 2 4小时。结论　建立的逆转录病毒介导的GM- CSF基因转移系统安全、有效 ,为 GM- CSF应用于肿瘤基因治疗提供了实验基础。Objective To provide an effective GM CSF gene transferring vector mediated by retrovirus and a basis for study of GM CSF gene modified tumor cell vaccines. Methods The recombinant retroviral vector pDORGM was constructed by cloning the GM CSF cDNA into the replication defective retroviral vector pDORneo, and transferred into packaging cell line PA317 by Lipofectin method. The viral titer was determined with the NIH3T3. Using the NIH3T3 amplification assay, replication competent retroviral particles were detected. A high titer of retrovirus was used to infect the human breast cancer cell line MCF 7. Results The retrovirus containing GM CSF gene had a highest titer of virus of 1×10 6 colony forming units/ml. No replication competent retrovirus were detectable in the vector preparation from the packaging cells. GM CSF gene modified MCF 7 cell lines stably released 500 to 800 units/10 6 cell of GM CSF within 24h in vitro. Conclusion The GM CSF gene transferring vector mediated by retrovirus was effective and safe. These results provied a basis for study of GM CSF gene used in cancer gene therapy.

关 键 词：GM-CSF 基因表达 乳腺肿瘤 基因转移 

分 类 号：R737.902[医药卫生—肿瘤]