逆转录病毒介导的肿瘤坏死因子基因转染的肿瘤细胞克隆株的建立及其生物学活性的观察

Observation on the Biological Characteristics of the Tumor Cells Transfected with Tumor Necrosis Factor-α Gene by Retrovirus

以逆转录病毒为载体将小鼠肿瘤坏死因子(TNF-α)基因转染人小鼠黑色素瘤细胞(B16)中,经G418抗性筛选,有限稀释及培养上清中TNF活性测定,从多株阳性克隆中筛选到一株高分泌TNF的克隆株(B16-TNF_a^(+)).B16-TNF_a^(+)细胞的形态,体外增殖能力及集落形成能力与野生型B16细胞无显著差异.体内致瘤性实验表明,B16-TNF_a^(+)细胞致瘤性下降,但致瘤性高低与接种细胞数量有关.小鼠皮下接种1.25×10~4或更多细胞,肿瘤结节形成率与接种细胞数量成负相关;而接种6.25×10~3细胞的小鼠肿瘤结节形成率反而高于2.5×10~4细胞接种的小鼠.接种B16-TNF_a^(+)的荷瘤小鼠长期存活率显著高于对照组.本实验为进行肿瘤细胞靶向的TNF基因治疗奠定了实验基础.

The in vivo tumorigenicity of murine B16 melanoma cells engineered to secret TNF-a was observed. The retrovirus containing mouse TNF-a cDNA was generated by the virus-packing cell PA317 transfected with plasmid pXT-TNF. The B16 cell clone secreting the highest TNF-a level was obtained after G418 resistance selection, limiting dilution and the assay of TNF-a activity. After the mice were inoculated subcutaneously with the cell clone, we found the tumor growth was inhibited and the survival period of the mice extended when compared with the mice inoculated with the wild-type B16 cells . We also found that the tuinorigenicity of B16-TNF-a+ cell was associated with the cell number inoculated. At or above the 1.25× 104 cells, the percentage of the mice with detectable tumor correlated negatively with the cell number inoculated: however, at the 6.25 × 103 cells, the percentage was higher than that at 2.5×10^(4) cells. These results encourage us to do further experiments on the following tumor cell-targeted TNF-a gene therapy.