携带人FL、GM-CSF基因的裸DNA的抑瘤作用

Antitumor gene therapy mediated by naked DNA as a carrier of human FL and GM-CSF gene

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.