肝癌血清特异糖链与癌组织中GnT-Ⅴ表达的相关性研究

摘要

目的：通过研究肝癌患者手术前、后血清中N-糖链谱图的变化来发现肝癌患者血清中的特异性糖峰，期望找到肝癌特异性血清标志物，并通过肝癌患者的临床癌组织及癌旁组织的GnT-V的表达来分析肝癌患者血清特异性糖峰与GnT-Ⅴ表达的相关性。方法应用基于DNA测序仪的荧光糖电泳（DSA-FACE）技术比较分析40例肝癌患者手术前、后血清及正常健康人血清中N-糖链谱图，并采用免疫组织化学技术检测40例肝癌患者的临床癌组织和癌旁组织中GnT-Ⅴ的表达。结果通过对血清N-糖链的DSA-FACE分析，确定了肝癌患者血清N-糖链谱图。选取在术前肝癌患者组显著高于术后肝癌患者组和健康人群组的糖峰中差异最显著的峰9（P＜0.001）以及术前肝癌患者组显著低于术后肝癌患者组和健康人群组的糖峰中差异最为显著的峰3、峰6（P＜0.05）作为特异性糖链，并以log（P9/P3*P6）为指标进行ROC曲线分析，发现曲线下面积为0.811±0.034，此指标检测的特异性为67%，灵敏度高达84%。运用免疫组化技术证实负责β-1,6分支的N-乙酰氨基葡萄糖转移酶Ⅴ（GnT-Ⅴ）在临床肝癌组织中的阳性率高达65%，而在癌旁组织中的阳性率仅有7.5%，差异具有统计学意义（P＜0.001）。结论糖峰9、3、6有可能成为辅助的肝癌早期诊断标志糖链，β-1,6分支糖链的变化主要与GnT-Ⅴ在癌症组织中高表达有关。%Objective The change of the glycan structure of glycoprotein is closely related to the occurrence and development of cancer. The project is aimed to find the glycan which is specific to hepatocarcinoma patients.The research result has high academic value in clarifying the relation between the different phases of cancer and the law of structure change of specific glycan in serum protein N-Glycans of hepatocarcinoma patient. And the function of the glycan in disease will be further defined. Methods To find Glycomic specific tumor markers of hepatocarcinoma, DSA-FACE was performed in 40 preoperation serum and post operation serum of hepatocarcinoma patients and 50 healthy human. And the same 40 primary hepatocarcinoma tissues and cancer nearby tissues were detected for the expression levels of GnT-Ⅴwith immunohistochemistry staining. Results The serum N-glycan profiles of hepatocarcinoma was identified by the DSA-FACE technique. The results indicated that there were significant differences in N-linked glycans between hepatocarcinoma patients and healthy persons. The Peak 3,6 and Peak 9 were the most significant peaks which were picked as a new tumor marker of hepatocarcinoma. Taking log (p9/p3*p6) as indicator of the ROC curve analysis, the area under ROC curve was 0.811±0.034 with 84%sensitivity and 67%specificity. The positive expression of N-acetylglucosaminyltransferase V(GnT-Ⅴ), which adds N-acetylglu-cosamine (GlcNAc) to the mannose of the trimannosyl core in aβ-1,6 linkage, in clinical cancer was 65%which is much higher than that in cancer nearby tissues 7.5%(P<0.001).Conclusion The Peak 9, 3 and Peak 6 were the most significant peaks which were picked as a new tumor marker of hepatocarcinoma. And the changes of theβ-1,6 branches was mainly caused by the expression of GnT-Ⅴin the prime hepatocarcinoma tissues.