转入人肿瘤坏死因子α基因的鱼腥藻IB02(+)的细胞毒活性及其体内抗肿瘤药效学研究

Cytotoxic and anti-tumor activities of transgenic Anabaena IB02(+) encoding hTNF-α

目的测定转入人肿瘤坏死因子α(hTNFα)基因的鱼腥藻IB02(+)的抗肿瘤药效。方法在相同的培养条件下通过100L光生物反应器培养4组转入hTNFα基因鱼腥藻IB02(+),分别命名为批次1、2、3、4,经过反复冻融和真空冷冻干燥处理,得到转入hTNFα基因鱼腥藻IB02(+)粗提物干粉样品。采用结晶紫染色法检测转入hTNFα基因鱼腥藻IB02(+)粗提物干粉样品的细胞毒活性,并通过测定不同批次间的细胞毒活性差异检测其稳定性。采用转入人肿瘤坏死因子α(hTNFα)基因鱼腥藻IB02(+)对小鼠肝癌细胞H-22的抑制来测定体内抗肿瘤药效。结果转入hTNFα基因鱼腥藻IB02(+)的细胞毒活性约为8000U/mg。4种不同批次的转入hTNFα基因蓝藻IB02(+)细胞毒活性相当,hTNFα蛋白性质比较稳定。小鼠1次口服最大耐受量为16.0g/kg,在观察期内所有受试小鼠无一死亡,生长发育正常,主要脏器未见异常,未显示明显毒性。药效学研究表明,口服6g/kg转入hTNFα基因蓝藻IB02(+)对小鼠肝癌细胞H-22的抑制率平均值(37.7±3.4)%,最高值为41.5%(P<0.001)。结论转入hTNFα基因鱼腥藻IB02(+)抗肿瘤作用明显,无明显的毒副作用且药效学性质稳定。

Objective To determine the anti-tumor effect of transgenic Anabaena IB02（＋） encoding hTNF-α. Method Four groups of （groups 1-4） transgenic Anabaena IB02（＋） encoding hTNF-α were cultured by 100 L photobioreactor under same conditions, and then treated with repeated freezing and thawing and cryodesiccation. L929 cell/crystal violet staining were employed to determine the in vitro cytotoxicity of the powers of transgenic Anabaena IB02（＋） encoding hTNF-α, and then the cytotoxicity of the four groups were compared to detect the stability of the method. The inhibitive effect of the powders were on mice transplantation tumor H-22 was determined to identify its pharmacodynamic activity in vivo. Results The cytotoxic activity of transgenic Anabaena IB02（＋） encoding hTNF-α was 8000 U/mg, which was almost the same in different groups, showing that the hTNF-α protein was stable. The maximum tolerated dose of oral administration in mice was 16.0 g/kg at one time. No mouse died during the observation period. The growth and development of the experimental mice were normal, and their main organs didn＇t show any abnormality, indicating that the powder was not toxic. The average inhibition rate of the mice transplantation tumor H-22 was （37.7 ± 3.4）% at a oral dose of 6 g/kg of transgenic Anabaena IB02（＋） encoding hTNF-α. Under the same condition, the highest value was 41.5% （P 〈 0.001）. Conclusion The transgenic Anabaena IB02（＋） encoding hTNF-α shows anti-tumor function and stable pharmacodynamic characteristics without obvious side effects.