This work was supported by a grant from The National Natural Science Foundation of China (20672144)
利用凝集素糖链特异亲和原理构建对细胞膜表面糖链进行即时检测的凝集素芯片体系,检测肝癌发生过程中细胞膜糖链的变化.从H22细胞系、正常小鼠和肝癌模型鼠肝组织中提取细胞进行荧光标记,激光扫描仪检测凝集素位点捕获的细胞,根据凝集素特异亲和性确定细胞膜表面糖表达谱,显微镜下观察捕获细胞的形态.对凝集素芯片捕获细胞的最佳条件进行探讨,用甘露糖抑制试验、流式细胞仪和不同血型红细胞验证了凝集素捕获细胞的特异性.结果显示:正常和肝癌小鼠肝细胞膜表面糖链存在较大差异,正常组只有PSA、DSL、STL、NPL凝集素位点捕获到细胞,实验组只有LTL和DBA位点没有捕获到细胞,提示小鼠肝癌组织细胞膜表面糖链显著增加,细胞膜上唾液酸、乙酰葡萄糖、乙酰半乳糖、甘露糖和半乳糖糖链表达增加,这些糖链及其相关糖蛋白可能在肝癌的发生和发展中起一定作用.该凝集素芯片有较好的稳定性和特异性,可以对细胞膜表面糖链进行动态、即时、通量的检测,为研究细胞膜表面聚糖在细胞发育和癌变等过程中的变化提供了一个技术平台.
To obtain information of glycan changes on cell surface in hepatocarcinoma progress, a high throughout lectin microarray was established to detect glycans on cell surface based on the principle of lectin to glycan binding affinity. Cells extracted from normal or model mice tissues and H22 cell lines were labeled with fluorescence and caught by lectins through the distinctive binding specificities, bound cells were directly detected by a laser fluorescence scanner to obtain the glycome profile of the cell surface according to the distinctive binding specificities of lectins on microarray and the appearance of bound cells were observed under the microscope. The optimal blocking buffer, optimal incubation time and temperature, optimal cell concentration were studied and specificity of lectin microarray was validated through the mannose blocking assay, flow cytometry and diffenrent blood type erythrocyte. High level of diversity of glycoprofiling was present between normal and hepatocarcinoma mice, only PSA, DSL, STL, NPL captured cells in normal group, all lectin captured cells except LTL and DBA in hepatocarcinoma group , the result show that Sia, GluNAc, GalNAc, Man and Gal increased on hepatocarcinoma cell surface. These results imply that these carbohydrates and correlate glycoprotein may play key roles in occurrence and development in liver canceration. The lectin microarray established a stable, real-time and throughout method and provides a novel strategy for study profiling changes of the cell surface glycome on tumor metastasis.
何群,李春辉,潘忠诚,王天骄,张玉魁,钟连声,王绍成,赵雨杰.应用凝集素芯片检测肝癌细胞膜表面糖链变化[J].生物化学与生物物理进展,2010,37(3):269-277
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