摘要
目的制备放射性药物99Tcm-annexinV,评估其在早期预测肺癌化疗效果中的作用。方法通过毕赤酵母重组表达、硫酸铵沉淀和分子筛层析纯化得到annexinV,采用氯化亚锡还原法在annexinV的氨基端标记放射性核素99Tcm,经脱盐柱纯化获得标记产物。采用薄层层析测定99Tcm-annexinV的标记率和放射性化学纯度,外露磷脂酰丝氨酸的红细胞测定99Tcm-annexinV的生物活性。通过在615小鼠腋部皮下接种LA795细胞和组织插块法获得荷肺癌小鼠模型,经腹腔注射环磷酰胺治疗肺癌后6、12、24和48h测定99Tcm-annexinV在小鼠体内的分布情况。结果毕赤酵母工程菌株分泌表达annexinV,经硫酸铵分级沉淀和分子筛层析纯化后annexinV得以有效回收。室温下30min完成anne-xinV的99Tcm标记,标记率为50.2%。99Tcm-annexinV放射性化学纯度为93.9%,其生物活性保存良好。生物分布实验表明,99Tcm-annexinV通过肾脏排泄。615荷肺癌小鼠模型经过环磷酰胺化疗后48h,99Tcm-annexinV在肿瘤组织的摄取达到最高,肿瘤/肌肉放射性摄取率之比为6.34,肿瘤/血液放射性摄取率之比为4.09。结论制备了毕赤酵母来源的99Tcm-annexinV,有可能应用于早期预测肺癌化疗效果。
Objective To prepare 99Tcm-annexinV and evaluate its application in the early prediction of therapeutic effect of chemical agent on tung cancer. Methods Annexin V was obtained by recombinant pichia pastoris expression, ammonium sulfate precipitation, and size-exclusion chromatography. The purified protein was labeled with 99Tcm at the N-terminal site by stannous chloride reduction and purified by desahing. The labeling yield and radiochemical purity of 99Tcm-annexinV were determined by instant thin-layer chromato- graphy. The biological activity of 99Tcm-annexinV was tested by phosphatidylserine-exposed erythrocytes bound radioactivity counting. The lung cancer mice models were established by inoculating LA795 cells to right flank of 615 mice subcutaneously and tumor tissue transplantation. The biodistribution of 99Tcm-annexinin V in lung cancer mice models were investigated at 6, 12, 24, and 48 h after eyclophosphamide administration. Results The annexin V was secreted from pichia pastoris and purified by ammonium sulfate precipitation and size-exclusion chromatography with high yield. The annexin V could be labeled at room temperature with 50.2% radioactivity yield. The radiochemical purity of 99Tcm-annexinVreached up to 93.9% with intact biological activity. The biodistribution analysis demonstrated that 99Tcm-annexinV was excreted from kidney. The uptake of 99Tcm-annexinV at tumor reached maximum 48 h after cyclophosphamide administration while tumor to muscle ratio was 6.34 and tumor to blood ratio was 4.09. Conclusions 99Tcm-annexinV derived from pichia pastoris was successfully prepared. It is useful in predicting the therapeutic effect of chemical agent on lung cancer.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2008年第5期595-598,F0003,共5页
Acta Academiae Medicinae Sinicae
基金
霍英东肺癌专项基金~~
