目的 开发一种能准确、特异地评价目标化合物对人氨肽酶N的抑酶活性检测试剂盒.方法 验证试剂盒准确度、精密度、稳定性等性能指标,同时通过与同类的传统试剂盒进行米氏常数的评价确认该试剂盒的灵敏度.结果 精密度验证,试剂盒的CV=4.29％符合CV<15％的要求;准确度验证中,标准化残差δ*=1.747落在(-2,2)区间以内,在95％置信度内是正常实验点,数据准确可靠;酶源稳定性良好;线性范围宽,96孔板中氨肽酶N在加入量120μL时还能呈现良好的线性关系;该试剂盒米氏常数低于传统试剂盒,说明灵敏度高.结论 所开发的试剂盒主要的性能指标表现良好,能准确测定化合物对人源氨肽酶N的抑酶活性,其检测成本和可靠性优于现有上市产品,具有推广应用及开发价值.

Breast cancer is a common type of cancer in females. Our previous studies indicated that leucine aminopeptidase 3 (LAP3) promotes migration and invasion of breast cancer cells. Vimentin is a mesenchymal marker, and its upregulation represents the promotion of epithelial–mesenchymal transition. In this study, we found that LAP3 and vimentin were highly expressed in breast cancer tissues, and the overexpression of LAP3 in breast cancer cells promoted the expression of vimentin. Western blot analysis indicated that the overexpression of LAP3 upregulated the phosphorylation of Erk1/2. MEK inhibitor PD98059 downregulated the expression of vimentin, matrix metalloproteinase-2/9 (MMP-2/9), and fascin through the inhibition of Erk1/2 activity. We hypothesized that LAP3 promoted tumor migration and invasion by upregulating vimentin. The knockdown of vimentin resulted in the inhibited migration and invasion of MDA-MB-231 and MDA-MB-468 cells. The expression of MMP-2/9 and fascin could also be downregulated. In conclusion, vimentin might play an important role in the promotion of breast cancer metastasis by LAP3.

Aminopeptidase N, also known as CD13, is a transmembrance protease with many functions. CD13 is involved in inflammatory diseases and cancers. A convenient and reliable laboratory test method for detecting the suppressing effects of enzyme activity would be useful for study of CD13 inhibitors. Porcine CD13 (pCD13) was traditionally considered an enzyme source but has significant practical disadvantages. pCD13 is not a human source, and the accuracy and reliability of experimental results are greatly reduced. In this study, a modified detection method with K562-CD13 monoclonal cells, a human-derived cell line, was established to detect the suppressing effects of enzyme activity by the CD13 inhibitor. In this method, K562-CD13 monoclonal cells were used as enzyme source and L-leucine p-nitroaniline hydrochloride as substrate. Using CD13 enzyme activity analyses, we found that the ability of the catalytic substrate was weaker in K562 cells than in the other cell lines, and K562-CD13 cells expressed significantly higher levels of CD13 enzyme activity than parental K562 cells. The enzyme activity of CD13 was detected with the new method after ubenimex treatment. The enzyme activity was significantly inhibited by ubenimex in a dose-dependent manner. In summary, this study proposes a sensitive, stable, and objective laboratory method for detecting the inhibitory effect of the CD13 inhibitor.