摘要
目的寻求诊断膀胱癌的检测新方法。方法收集8例膀胱癌患者术前和术后3个月(正常)尿液标本16份,提纯纤维连接蛋白(Fn),辣根过氧化物酶(HRP)标记的凝集素作探针,连接到已固定在膜上的Fn糖链,检测已结合的HRP代表Fn糖链与凝集素的结合力,增强化学发光法(ECL)检测已结合HRP的结合力。高效液相(HPLC)和荧光标记底物测定膀胱癌和癌旁正常组织N-乙酰氨基葡萄糖转移酶(GnT)活力。结果用伴刀豆球蛋白凝集素(ConA)-HRP作探针时,膀胱癌尿液Fn结合力仅为正常膀胱尿液Fn的0.18,而用蔓陀罗凝集素(DSA)-HRP和麦胚凝集素(WGA)-HRP作探针时,膀胱癌组的结合力为正常组的3.34倍和3.26倍。膀胱癌患者尿液中FnN-糖链的天线数和平分型GlcNAc结构都升高。尿Fn与WGA的结合力与病理分期、分级相关。膀胱癌组织GnT-Ⅲ、Ⅳ和Ⅴ的酶活力比癌旁正常组织分别增加34.0、18.1和1.6倍。结论凝集素-HRP检测分析尿液Fn的N-糖链结构改变有可能是一种简单和精确诊断膀胱癌的方法。
Objective To investigate a new method for diagnosis of bladder cancer （BCa）. Methods Sixteen urine samples from 8 patients with BCa preoperatively and 3 months postoperatively （proved to be normal） were collected, and the fibronectin （Fn） in the urine samples were purified. Different lectins labeled with horseradish peroxidase （HRP） were used as probes to bind the glycans of purified Fn immobilized on membrane. The enhanced chemiluminescence （ECL） reagent were adopted to estimate the activity of the bound HRP as a measure of the binding affinity of the Fn glycans to lectins, and expressed as luminescent light units （LLU）. The enzymatic mechanism of the structural alteration of N-glycans in BCa Fn was studied with determination of GnT activities using HPLC method and fluorescent labeled substrate. Resuits The mean value of LLU of BCa Fn was only 18.1% of the normal samples when Con A-HRP was used as probes, while the mean LLU of BCa samples were 3.34 times and 3.26 times of the normal for the probes of DSA-HRP and WGA-HRP, respectively. These results revealed that the antennary number and bisecting GlcNAc structure were increased in the N-glycans of urine Fn from BCa, Besides, the binding affinity of Fn with WGA was correlated with pathological stage and grade, The results of GnT determination showed that GnT-Ⅲ, Ⅳ and Ⅴ in BCa tissues were increased by 34.0, 18.1 and 1.6 times respectively of the normal bladder tissues, Conclusions The lectin-HRP assay for analyzing the structure of N-glycans in urine Fn may be used as a simple and accurate diagnostic method for BCa in the future.
出处
《中华泌尿外科杂志》
CAS
CSCD
北大核心
2005年第9期601-604,共4页
Chinese Journal of Urology
基金
吴阶平泌尿外科基金资助项目(962132)
