摘要
目的探讨可表达人FL和GM-CSF的裸DNA抑制小鼠移植肿瘤生长的作用.方法分别将带有信号肽序列的人FL和GM-CSF基因插入pcDNA3.1/zeo载体,构建分泌表达人FL和GM-CSF的治疗用表达载体,经Sephacryl S1000柱层析法纯化了重组表达质粒.用Western 印迹和ELISA法检测目的基因在293细胞中的表达,并用TF1细胞验证293细胞表达的GM-CSF的生物学活性.重组表达质粒与脂质体孵育形成复合物,隔天肌肉注射小鼠共6次,每只小鼠接种T细胞淋巴瘤细胞EL-4 2×105后,观察不同基因组合条件下肿瘤的生长情况.结果人FL和GM-CSF在293细胞中表达量分别为50 μg/L和30 μg/L,单用FL或GM-CSF和两者联用对肿瘤生长均有一定抑制作用,在第17天抑制率为30%~40%.联合应用组较单用FL或GM-CSF组更为有效抑制肿瘤生长,但小鼠生存时间并无延长.结论人FL和GM-CSF在293细胞中均获表达,且对肿瘤的早期生长具有一定的抑制作用,两者联合应用效果更为明显,但对小鼠最终生存时间并无影响.
Objective This article investigated the antitumor effects mediated by naked DNA coding human FL and GM-CSF. Methods Two recombinant vectors containing human FL or GM-CSF genes, hFL-pcDNA3.1/Zeo and hGM-CSF-pcDNA3.1/Zeo, were constructed. A large scale plasmid DNA production and purification were followed by a size exclusion chromatography, Sephacryl S1000. The two recombinant plasmids and pcDNA3.1/Zeo were introduced into 293 cells, and the product was collected and tested by Western blotting and ELISA. The expressed hGM-CSF stimulated the proliferation of TF1 cells in vitro. A stable transfection complex was prepared by mixing plasmid DNA with a cation liposome, Lipofectamine 2000 and delivered to C57BL/6 mouse intramuscularly every second day. After a total of six injections, 2 ×10^5 EL-4 cells, a murine T-lymphoma (thymoma) cell line was inoculated into these mice subcutaneously and the antitumor effect of the complex was evaluated every two days. Results The expression amounts of FL and GM-CSF in 293 cells were 50μg/L and 30μg/L, respectively. Treatment with FL or GM-CSF alone and combination treatment of both factors can suppress the growth of the tumor between 30% - 40%. Conclusion The mice received expression vectors can suppress the growth of the tumor comparing with pcDNA3.1/Zeo, combinated application of both expression vectors showed more efficient antitumor effects. However, suppression of the growth of tumors failed to increase surviving days of these mouse.
出处
《基础医学与临床》
CSCD
北大核心
2005年第10期914-919,共6页
Basic and Clinical Medicine
基金
国家高技术研究发展计划项目(863项目)(2001AA212171)