苦荞凝集素对结肠癌细胞增殖的抑制作用

采用醋酸铵缓冲液抽提、硫酸铵分级沉淀、阴离子交换和分子排阻层析等方法，首次从苦荞麦中提取出一种苦荞凝集素(tatary buckwheat lectin，TBL). MTT检测发现，TBL对3种结肠癌细胞 DLD-1、HCT116及SW480的增殖有显著的抑制作用，且呈剂量依赖效应，而对正常细胞及其它类型癌细胞的增殖无明显影响. 采用兔网织红细胞裂解系统和荧光素酶检测系统检测TBL对蛋白质合成的影响，结果表明，随着TBL浓度的增大，蛋白质翻译抑制作用逐渐增强，当TBL浓度为40 μg/mL时荧光素酶活性下降至50%. 另外，TBL与兔网织红细胞裂解系统作用后，核糖体RNA出现新的片段，表明TBL具有N-糖苷酶的活性. 将HCT116细胞总RNA与TBL体外作用后,发现二者存在明显的相互作用，核糖体RNA被降解. qRT-PCR检测显示，TBL明显下调HCT116细胞中多种microRNAs (miRNAs) 表达. 综合以上实验得出，TBL是一种Ⅱ型核糖体失活蛋白(type-Ⅱ ribosome-inactivating protein, Ⅱ-RIP)类的植物凝集素，具有N-糖苷酶活性，可下调肠癌细胞中多种miRNAs的表达，抑制结肠癌细胞增殖.

Proliferative Inhibition of Tatary Buckwheat Lectin in Colon Cancer Cells

A plant lectin, tatary buckwheat lectin (TBL), was purified from tatary buckwheat seeds by procedures of ammonium acetate extraction, ammonium sulfate fractionation, ion exchange and molecular exclusion chromatography. Dose-dependent inhibition of TBL on the growth of DLD-1, HCT116 and SW480 cells were found by MTT assays. Furthermore, TBL had no proliferative inhibition on normal cells and other types of cancer cells. The influence of TBL on protein synthesis was detected using both rabbit reticulocyte lysate system and luciferase system. The results showed that the inhibition of protein translation was intensified as the TBL concentration increased. The luciferase activity reduced to about 50% at the TBL concentration of 40 μg/mL. In addition, fragmented ribosomal RNA was detected, suggesting that TBL was likely to exhibit a N-glycosidase activity. An interaction between TBL and in vitro RNAs might exist in the rRNA degradation in HCT116 cells. The qRT-PCR analysis showed that certain miRNAs were down-regulated in TBL-treated HCT116 cells. In conclusion, the new lectin from tatary buckwheat seeds was indicated as a type-Ⅱ ribosome-inactivating proteins(Ⅱ-RIP) with a N-glycosidase activity. TBL might be able to down-regulate the expression of certain miRNAs to inhibit the growth of colon cancer cells.