羟乙基壳聚糖抗人死亡受体5单链抗体纳米粒的制备、鉴定及体内抑瘤研究

Preparation and identification of aDR5ScFv loaded GCS and assaying its anti-tumor effect in vivo

目的为提高抗人死亡受体5单链抗体治疗肝癌的效果,探讨羟乙基壳聚糖抗人死亡受体5单链抗体纳米粒(GCS-aDR5ScFv)的制备、鉴定并研究其对小鼠肝癌H22模型的治疗效果及机制。方法用亲和镍柱层析法纯化抗人死亡受体5单链抗体(aDR5ScFv),采用离子凝胶法制备GCS-aDR5ScFv,应用扫描电镜来检测GCS-aDR5ScFv的外观形态,激光粒度分析仪分析纳米粒的粒径及粒径分布,并检测其表面Zeta电势。建立小鼠肝癌H22模型,用0.272 mg/ml GCS-aDR5ScFv隔天治疗并测量小鼠体质量及肿瘤大小,治疗2周。通过Western blot检测active-caspase8、active-caspase3及BAX表达。结果纯化的aDR5ScFv符合理论条带(Mr30 000),GCS-aDR5ScFv纳米粒形态均一,纳米粒径大小、Zeta电势和多分散指数表明GCS-aDR5ScFv稳定。与正常组相比,治疗组小鼠体质量显著差异,治疗组肿瘤体积和大小差异显著。Western blot检测active-caspase8、active-caspase3、BAX的表达水平上调。结论本研究制备的GCS-aDR5ScFv稳定,对小鼠肝癌H22模型具有抑瘤作用,其机制可能与active-caspase8、active-caspase3及BAX蛋白表达上调相关。

In order to improve the effect of anti-human death receptor 5 single-chain antibodies in the treatment of liver cancer, we assayed the therapeutic effect and mechanism of GCS-aDRsScFv therapy in mouse hepatoma 22 （H22） model. GCS-aDRsScFv was prepared and confirmed by electron microscopy and laser particle size analysis instrument, then used to immunize H22 mouse every other day at dosage of 0.272 mg/ml for 2 weeks. We found that the body weight was improved but the tumor volume/weight decreased in immunized mice as compared with control mice. Furthermore, Western blot showed that GCS-aDR5ScFv treatment could elevated the expression levels of active-caspase8, active-caspase3, and BAX in H22 mice. In conclusion, GCS-aDR5ScFv posses anti-tumor effects in H22 mice, and its mechanism may related with upregulation of active-caspaseS, active-caspase3, and BAX protein expression.