凝集素芯片检测肿瘤标志物糖蛋白N-聚糖及其在诊断中的意义

Identification of N-glycan moiety of tumor marker by lectin microarray

背景与目的:肿瘤的发生、发展伴随着相关糖蛋白的糖基化改变,糖蛋白聚糖结构的多样性对其生理功能起着重要的作用。凝集素芯片是一种简单、快速、高通量的方法,可以分析肿瘤标志物糖蛋白的N-聚糖部分,是肿瘤相关糖蛋白质组学有力的研究工具。本研究探讨使用凝集素芯片检测肿瘤标志物糖蛋白N-聚糖,以期鉴别疾病性质。方法:利用生物芯片技术使用凝胶基片构建以不同凝集素和荧光标记糖蛋白特异性亲和为原理的凝集素芯片,可以检测糖蛋白的N-聚糖的特征性结构,比较相似糖蛋白的差异,进一步可以检测和比较不同来源相同肿瘤标志物糖蛋白的N-聚糖部分的细微差异。结果:凝集素芯片结果准确,荧光标记糖蛋白浓度与荧光值达10～1000nmol/L的Cy3浓度范围内存在线性关系,1mg/ml是合适的凝集素点样浓度;凝集素芯片非常敏感,在本研究的条件下,最低可测得达1pg的相应糖蛋白,凝集素芯片结合荧光探针技术可以鉴别不同来源甲胎蛋白N-聚糖结构的细微差别。结论:凝集素芯片检测糖蛋白N-聚糖结构切实可行、准确方便;凝集素芯片可以给出肿瘤标志物糖蛋白N-聚糖结构的信号模式图及其变化,在疾病的早期诊断方面具有重要意义。

Background and purpose： Most of the tumor markers are glycoproteins. The occurrence of cancer is accompanied by changes in glycosylation of related tumor marker. A simple, rapid and high-throughput manner for analyses of the N-glycan moiety of tumor marker is needed in the cancer glycoproteome study in order to identify the N-glycan moiety of tumor marker. Methods： Gel-slide was used as solid support of lectin microarray. The lectin microarray was fabricated with selected lectins to compare the N-glycan moieties of two similar glycoproteins and further to compare the N-glycan compositions of AFPs from different sources. Results： There was a linear relationship between the fluorescence intensity and the Cy3 concentration of glycoprotein at the range from 10 to 1000 nmol/L. The optimum loading concentration of lectins in this array is 1 mg/ml. The lectin microarray was sensitive and it could even detect glycoprotein 1 pg. The lectin microarray could indicate the minimal differences of N-glycan compositions between similar glycoproteins and could also indicate the minimal differences of N-glycan compositions between AFPs from different sources. Conclusion： The lectin microarray could be used to identify the N-glycan components of glycoprotein. Gel-slide is a good choice for the fabrication of lectin microarray. The lectin microarray could give a panel of lectin affinity patterns of individual glycoproteins. It can identify the ＂glyco-label＂ of disease marker, the lectin microarray is important in the early diagnosis of diseases including cancer. The developed lectin affinity microarray may contribute to the research of glycoproteomics associated with cancer.