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目的观察抗肝癌重组单链免疫毒素hdsFv-RC-RNase对荷人肝癌裸鼠移植瘤的导向治疗作用,并探讨其临床应用价值。方法将原核表达载体TIG-hdsFv-RC-RNase转化E.coliBL21(DE3)plys中,利用IPTG大量诱导表达抗肝癌hdsFv-RC-RNase重组单链免疫毒素。表达产物在非变性条件下经Ni-NTAAgarose亲合层析法纯化并体外复性,利用细胞ELISA法检测其特异性结合体外培养的人肝癌细胞的免疫活性。建立荷人肝癌裸鼠移植瘤动物模型,初步评估其对荷人肝癌裸鼠移植瘤的导向治疗作用。结果工程菌经IPTG诱导后,出现一条相对分子质量约为41000的新生蛋白带,且主要以可溶性形式表达。纯化后表达产物的纯度达到基本均一。细胞ELISA检测结果显示,纯化产物复性后能够与体外培养的人肝癌细胞特异性结合,而对正常肝细胞的结合能力非常弱,两者具有非常显著的差异(P<0.01)。导向治疗结果表明,其对荷人肝癌裸鼠移植瘤治疗有效率为100%,与生理盐水组相比具有非常显著的差异(P<0.01),抑瘤率达到79.38%。结论成功获得了特异性强的有活性的抗肝癌重组单链免疫毒素hdsFv-RC-RNase,其对荷人肝癌裸鼠移植瘤具有一定的导向治疗作用,可能成为抗肝癌导向治疗药物。
Objective To observe the anti-hepatoma recombinant single-chain immunotoxin hdsFv-RC-RNase on human hepatocellular carcinoma xenograft tumor-oriented treatment and to explore its clinical value. Methods The prokaryotic expression vector TIG-hdsFv-RC-RNase was transformed into E.coli BL21 (DE3) plys. IPTG was used to induce recombinant hSSFV-RC-RNase recombinant single chain immunotoxin. The expressed product was purified under non-denaturing conditions by Ni-NTA Agarose affinity chromatography and refolded in vitro. The cell-specific immunostaining of human hepatoma cells cultured in vitro was detected by ELISA. To establish animal model of human hepatocellular carcinoma xenograft in nude mice and to evaluate its effect on the induction therapy of hepatocellular carcinoma xenografts in nude mice. Results After inducing by IPTG, a nascent protein band with a relative molecular mass of about 41000 appeared mainly in soluble form. After purification, the purity of the expressed product was basically uniform. The result of cell ELISA showed that the renaturation of the purified product could specifically bind to human hepatocellular carcinoma cells cultured in vitro, while the ability of binding to normal hepatocytes was very weak (P <0.01). The results of targeted therapy showed that the effective rate of transplanted hepatocellular carcinoma in nude mice was 100%, which was significantly different from that of saline group (P <0.01). The tumor inhibition rate was 79.38%. Conclusion The specific and active anti-hepatoma recombinant single chain immunotoxin hdsFv-RC-RNase was successfully obtained. It has certain therapeutic effect on the transplanted tumor of human hepatoma in nude mice and may become the anti-hepatocarcinoma drug.