尿液蛋白质组在膀胱癌临床诊治中的应用

膀胱癌是一种常见的泌尿系统疾病,尿细胞学检查与膀胱镜检查是膀胱癌的主要临床诊断手段,但尿细胞学检查敏感性较差,膀胱镜检查为侵入性检查,易给病人带来强烈的不适感;且膀胱癌具有易复发的特点,大部分患者必须面临频繁的检查,临床亟需发展舒适、准确的检查手段.尿液存储是膀胱的主要生理作用,尿液可以直接接触肿瘤实体,肿瘤分泌的一些蛋白质分子极可能进入尿液中,并且患者尿液样本便于足量多次收集.同时,蛋白质组技术以及尿液蛋白质组研究的快速发展,为我们利用尿液研究膀胱癌提供了便利的途径.本文系统总结了尿液蛋白质组研究的主要技术手段,重点关注膀胱癌尿液蛋白质组研究趋势和应用方向,以期为利用尿液蛋白质组研究膀胱癌提供助力.     

膀胱癌尿液生物标记物的现状与展望

膀胱癌是世界上最常见的肿瘤之一,膀胱肿瘤的早期检测对改善病人预后至关重要。目前检测和监视膀胱肿瘤的常规方法是膀胱镜检查联合应用尿细胞学检查,但其局限性已经表明膀胱癌需要更好的诊断方法。理想中的诊断方法应该是非侵袭性的,既能够诊断,也能监测疾病的复发,并且能够为治疗提供依据。因此,以尿液为基础的非侵袭性方法将会给患者和医院带来福音。本文综述了近年来报道的膀胱癌尿液生物标记物及相关新技术。     

miR-126 在膀胱癌患者尿液中的表达及临床意义

目的:探讨miR-126在膀胱癌患者尿液中的表达与临床病理特征的关系,评估miR-126的肿瘤标志物诊断价值。方法:收集48例初发膀胱尿路上皮癌患者与32例健康对照者晨尿,提取尿液总RNA,通过实时荧光定量PCR技术检测各样本中的miR-126的表达水平,并经受试者工作曲线(ROC)分析其诊断价值。结果:膀胱癌患者尿液中的miR-126表达水平相对健康对照组明显上调(P0.01),其表达水平在不同病理级别之间存在显著差异(P均0.05),且低级别组表达水平略高于高级别组,与肿瘤大小、数目以及淋巴转移也有一定的相关性(P0.05),而与患者的年龄、性别、TNM分期等均无相关性(P0.05)。通过ROC曲线分析尿液中miR-126诊断膀胱肿瘤的曲线下面积(AUC)为0.861,当最佳切点定在7.475时,miR-126诊断膀胱肿瘤的敏感性和特异性分别为75.0%、81.2%。结论:膀胱癌患者尿液中miR-126的表达差异能够反映病情进展程度,其表达水平对膀胱肿瘤的早期诊断及病情评估具有一定的价值。     

肿瘤微环境中ROS介导IgG表达对膀胱癌细胞增殖和侵袭能力的影响

摘要 目的：探讨肿瘤微环境（TME）中活性氧（ROS）介导免疫球蛋白G（IgG）表达对膀胱癌EJ细胞增殖、迁移和侵袭能力的影响。方法：临床收集的18例膀胱癌患者样本,通过Western blot法检测膀胱癌和癌旁正常组织样本中IgG表达量。利用免疫荧光染色（IF）技术分别对膀胱癌组织和癌旁正常组织中ROS和IgG分子进行共定位和相对定量分析。将活性氧清除剂N-乙酰基-L-半胱氨酸（NAC）加入膀胱癌细胞EJ中,实验分为3组：空白组（EJ细胞）、阴性对照组（EJ+PBS）、实验组（EJ+PBS+NAC）,10 mM NAC药物处理48小时后,运用DHE-ROS荧光探针技术和Western blot实验检测药物NAC对ROS和IgG相对表达水平的影响;通过克隆集落形成实验、划痕实验、Transwell实验检测去除ROS后对膀胱癌细胞增殖、迁移和侵袭的影响。结果：人体膀胱癌组织中ROS和IgG分子表达水平显著高于癌旁正常组织（P<0.001）;荧光显微镜显示膀胱肿瘤组织中正常膀胱尿路上皮细胞组织被肿瘤细胞严重破坏,结构紊乱不规则,IgG和ROS表达水平均升高,而癌旁组织膀胱尿路上皮组织的结构均匀规则;NAC药物处理EJ细胞后,与空白组和阴性对照组相比ROS和IgG表达显著降低,同时实验组细胞的增殖、迁移和侵袭能力明显下降（P<0.01）。结论：ROS和IgG在临床膀胱癌组织和体外膀胱癌细胞株EJ中均显著高表达,在肿瘤微环境中ROS通过调控IgG表达,从而促进膀胱癌细胞的增殖、迁移、侵袭。ROS和IgG可能成为膀胱癌早期诊断和生物治疗的临床新靶点。     

基于FFPE组织切片的膀胱癌N-连接糖链原位酶解及分析

目的 研究膀胱癌FFPE组织切片的N-连接糖链,发现膀胱癌FFPE肿瘤组织的异常N-连接糖链修饰情况。方法 发展基于FFPE组织切片原位提取N-连接糖链的实验流程。通过PNGase F酶切FFPE组织解释放N-连接糖链。对N-连接糖链自由端进行全甲基化修饰。通过MALDI-TOF/TOF-MS检测N-连接糖链的相对含量。进行数据库匹配,确定N-连接糖链的可能糖型。ROC分析用于预测显著差异N-连接糖链作为预测膀胱癌生物标志物的准确度。结果 MALDI-TOF/TOF-MS检测泛甲基化修饰N-连接糖链的数据显示,在16例膀胱癌患者的肿瘤和癌旁组织的3次重复实验中,肿瘤组织中蛋白质高甘露糖型N2H6、N2H7、N2H8、N2H9和复杂型N5H6F1糖链修饰水平显著上升,同时高甘露糖型N2H5、杂合型N3H5以及复杂型N3H4、N4H4、N5H6F1S2糖链修饰水平显著下降。ROC分析显示,双天线型N-连接糖链N3H4（AUC=0.90）和N4H4（AUC=0.91）在单独或者共同区分膀胱癌患者肿瘤组织和癌旁组织中都具有很好的可靠性,可能成为膀胱癌的潜在生物标志物。结论 膀胱癌FFPE肿瘤组织中存在蛋白质异常N-糖基化修饰,N-连接糖链N3H4和N4H4或可成为膀胱癌的潜在生物标志物。     

膀胱癌组织CALD1 mRNA表达水平与临床病理特征和预后的关系分析

摘要 目的：探究膀胱癌组织钙调蛋白结合蛋白（CALD1）信使RNA（mRNA）表达水平与临床病理特征和预后的关系。方法：选取2015年7月-2017年7月于我院收治并确诊的111例膀胱癌患者为研究对象,收集癌组织及癌旁正常组织,采用逆转录聚合酶链式反应（RT-PCR）法检测CALD1 mRNA在膀胱癌组织与癌旁正常组织中的表达差异,分析CALD1 mRNA相对表达量与临床病理特征的关系。对患者进行5年随访,分析CALD1 mRNA相对表达量与预后的关系。结果：CALD1 mRNA在膀胱癌组织的相对表达量显著高于癌旁正常组织（P<0.05）;CALD1mRNA相对表达量与膀胱癌患者组织分化程度、TNM分期、淋巴结转移相关（P<0.05）;111例患者出院后随访5年,4例失访,107例患者获得随访,CALD1低表达组和高表达组的5年总生存率分别为72.34%（34/47）、50.00%（30/60）（P＜0.05）,CALD1低表达组5年总生存率高于高表达组;Cox比例风险回归模型分析显示,组织低分化程度、TNM高分期、淋巴结转移、CALD1mRNA高表达是影响膀胱癌患者预后的危险因素（P<0.05）。结论：CALD1在膀胱癌组织中呈高表达,CALD1mRNA相对表达量与患者组织分化程度、TNM分期、淋巴结转移及预后相关。     

尿液蛋白质组作为下一代生物标志物的潜力

生物标志物是与机体生理及病理生理状态相关的可监测到变化的生化指标,尿液不属于内环境,没有稳态机制,能够积累并反映机体生理状态的早期变化,有潜力辅助疾病的早期诊断和预后监测。得益于非侵入性的收集方式,尿液可以被连续、大量、重复收集并便捷、稳定地保存,且组分相对简单,易于分析,是理想的标志物研究样本。但临床尿液样本蛋白质组可能会受到生活习惯、用药情况等多种混杂因素的影响,而动物模型方便控制变量,可以最大程度减少混杂因素的干扰,并使得在疾病发生、发展极早期采集样本成为可能;此外,患者的疾病分期、分型、用药情况等信息不能被忽视,现有样本策略和分析方式有待优化,例如,对同一个人不同时期、不同状态(例如患病前后)的尿液样本进行前后对照是一种理想的分析方式,这种方式能够消除个体间差异性的影响,符合个性化、精准化医疗的趋势;在无自身对照样本的情况下,一对多的分析方法能够更好地体现个体与健康群体的差别,辅助未知疾病的诊断和鉴别。尿液大分子的膜保存方式使得临床样本的保存更加简单经济。尿液生物标志物领域研究的进步需要政策和伦理的支持、资金和人力长期持续的投入以及大样本、大数据的辅助。本文综述了尿液生物标志物...     

尿液作为新型生物标志物来源的探索

机体各种变化是生物标志物研究的核心内容.因为机体固有的稳态调控机制,在血液中早期产生的变化容易被清除.而在尿液中不存在稳态调控机制,允许容纳更多的变化因素存在.尤其在疾病发生的早期阶段,从尿液中更有可能发现新型的早期生物标志物.在尿液生物标志物研究中,亦应当考虑药物的影响.使用尿液生物标志物研究路线图,能够有效规避各种影响因素对于尿液生物标志物研究的干扰;同时,结合膜处理新技术,有利于经济、高效地大规模收集尿液样本,促进尿液生物标志物的大规模研究.另外,本文介绍了最容易在尿液中体现出变化的肾脏疾病生物标志物的研究进展.尿液生物标志物的研究,将赋予人类在疾病预防、诊断、治疗及预后监测等诸多方面实现更多进步的可能性.     

ELISA定量分析尿液BLCA-1/-4水平诊断膀胱癌的临床价值分析

目的:探讨膀胱癌患者尿液膀胱特异性核基质蛋白(bladder cancer specific nuclear matrix proteins,BLCA)-1/-4水平及其临床应用价值。方法:本研究纳入38例膀胱癌患者、40例正常对照组。采集受试者尿液样本,通过竞争性酶联免疫吸附法(enzymelinked immunosorbent assay,ELISA)定量分析尿液中BLCA-1和BLCA-4的水平,绘制受试者工作曲线,确定cut-off值。结果:膀胱癌患者尿液BLCA-1/-4水平均显著高于对照组(P0.001);当cut-off值取0.859 ng/mL时,BLCA-1诊断膀胱癌的敏感性和特异性分别为71%(27/38)、90%(36/40)。肌层浸润性膀胱癌患者尿液BLCA-1较非肌层浸润性膀胱癌患者水平显著升高(P0.001),但不同分级膀胱癌患者尿液BLCA-4水平无显著差异(P0.05)。高级别膀胱癌患者尿液BLCA-4水平较低级别膀胱癌患者显著升高(P0.05),但不同分期膀胱癌患者尿液BLCA-4水平无显著差异(P0.05)。以cut-off为0.859 ng/mL时,BLCA-1诊断膀胱癌的敏感性和特异性分别为71%(27/38)、90%(36/40)。以cut-off为0.620 ng/mL时,BLCA-4诊断膀胱癌的敏感性和特异性分别为76.3%(29/38)、97.5%(39/40)。联合检测尿液BLCA-1和BLCA-4诊断膀胱癌的敏感性和特异性分别为84.2%(32/38)和100%(40/40),准确度为0.923(77/78),阳性预测值为100%(32/32),阴性预测值为86.9%(40/46)以及YOUDEN指数分别为0.842。结论:膀胱癌患者尿液BLCA-1和BLCA-4水平显著升高,且敏感性和特异性均较高。联合检测尿液BLCA-1和BLCA-4较单一检测用于诊断膀胱癌的临床应用价值更高。     

近红外荧光染料IR-783在膀胱癌中的成像研究

目的:观察研究近红外荧光染料IR-783在膀胱癌中的特异性成像。方法:通过染料与膀胱癌细胞及正常细胞共孵育,观察近红外荧光染料IR-783是否能够实现膀胱癌细胞的选择性成像。利用细胞器示踪剂观察近红外荧光染料在膀胱癌细胞内的共定位;使用IR-783检测循环血液中及尿液中的膀胱癌细胞。结果:近红外荧光染料IR-783可被膀胱癌细胞选择性摄取。IR-783可选择性聚集在膜性细胞器如线粒体和溶酶体内,这种选择性聚集作用使IR-783可以保持较长的染色效果。近红外染料可以检测到血液或尿液极少量的膀胱癌细胞。结论:近红外荧光染料IR-783能够被膀胱癌细胞特异性吸收,可用于血液和尿液中膀胱肿瘤细胞的特异性诊断,具有重要的临床应用前景。     

Global changes in bladder cancer mortality in the elderly

BackgroundBladder cancer is the 14th most common cause of cancer deaths worldwide and has a mean age of diagnosis of 73 years. Elderly people have fewer curative treatment options for muscle invasive bladder cancer. The aim of this study is to investigate how bladder cancer mortality has changed over the past forty years in different world regions to assess discrepancies between elderly and younger patients with bladder cancer.MethodsBladder cancer mortality data were extracted from the World Health Organisation’s GLOBOCAN database. Age-standardised mortality rates (ASMR) for bladder cancer were computed by year, sex, region and Human Development Index (HDI) using the world standard population.ResultsOverall ASMR in all available countries with data between 1986 and 2014 for men aged ≥ 75 has decreased from 101.2 to 89.9 per 100,000 (−11.2%). The decrease in ASMR for men < 75 has been 0.3–2.0 per 100,000 (−39.4%). In women aged ≥ 75 ASMR has decreased from 26.9 to 22.5 per 100,000 (−16.4%) and in women < 75 the ASMR has decreased from 0.76 to 0.56 per 100,000 (−26.4%).Correlation analysis showed a positive linear relationship between Human Development Index (HDI) and improvement in age-standardised mortality rate in all ages. Pearson’s coefficient showed that correlation was strongest in the 60–74 age group (r = −0.61, p < 0.001) and weakest in those aged ≥ 75 (r = −0.39, p = 0.01).ConclusionBladder cancer mortality is not improving in the elderly at the same rate as the rest of the population. Particular focus should be applied in future research to enhance and expand treatment options for bladder cancer that are appropriate for elderly patients.     

BLCA-4在膀胱癌中作用的研究进展

近年来,膀胱癌的发病率与死亡率呈逐年上升,已成为泌尿系统最常见的肿瘤。随着分子生物技术的不断发展,核基质蛋白（SMVs）、膀胱肿瘤抗原（BTA）、透明质酸和透明质酸酶（HA—Haase）、细胞角蛋O（CK）等诸多膀胱肿瘤标志物被发现。其中,膀胱癌特异性核基质蛋白-4（BLCA-4）是一种可溶性的复合物,在正常膀胱组织中不表达而存在于膀胱癌组织中。检测尿样中BLCA-4水平有望用于膀胱癌的筛查及诊断,具有较高的敏感性和特异性,也可用于普通人群与膀胱癌高危人群（脊髓损伤患者）的筛选与检测;而术后病理组织检测BLCA-4的表达也可为膀胱癌患者的预后预测提供参考。本文对近年来BLCA-4在膀胱癌中的作用进行了综述。     

BLCA-4 在膀胱癌中作用的研究进展

摘要：近年来,膀胱癌的发病率与死亡率呈逐年上升,已成为泌尿系统最常见的肿瘤。随着分子生物技术的不断发展,核基质蛋白 (NMPs)、膀胱肿瘤抗原(BTA)、透明质酸和透明质酸酶(HA-HAase)、细胞角蛋白(CK)等诸多膀胱肿瘤标志物被发现。其中,膀胱癌 特异性核基质蛋白-4(BLCA-4)是一种可溶性的复合物,在正常膀胱组织中不表达而存在于膀胱癌组织中。检测尿样中BLCA-4 水平有望用于膀胱癌的筛查及诊断,具有较高的敏感性和特异性,也可用于普通人群与膀胱癌高危人群(脊髓损伤患者)的筛选与 检测;而术后病理组织检测BLCA-4 的表达也可为膀胱癌患者的预后预测提供参考。本文对近年来BLCA-4 在膀胱癌中的作用进 行了综述。     

Discovery of Apo-A1 as a potential bladder cancer biomarker by urine proteomics and analysis

Bladder cancer is clinically characterized by high recurrent rate and poor prognosis and thereby patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method for detecting and monitoring bladder cancer would thus be advantageous. In this study, by using the two-dimensional electrophoresis (2-DE) approach with subsequent mass spectrometry (MS), we demonstrated the increased expression of apolipoprotein-A1 (Apo-A1) in individual urine from patients with bladder cancer, which was confirmed by Western blot results. A further analysis of the urinary Apo-A1 levels by an enzyme-linked immunosorbent assay yielded results that were consistent with the Western blot, and suggested Apo-A1 could provide diagnostic utility to distinguish patients with bladder cancer from healthy controls at 19.21 ng/ml. Further validation assay in a larger number of urine samples (n = 379) showed that Apo-A1 could be used as a biomarker to diagnosis bladder cancer with a sensitivity and specificity of 89.2% and 84.6% respectively. Moreover, the application of exfoliative urinary cytology in combination with the urine Apo-A1 detection could significantly increased the sensitivity in detecting bladder cancer. Our data showed a significant relationship of expressed Apo-A1 was established between bladder cancer and normal controls. Apo-A1 could be a potential biomarker for the diagnosis of bladder cancer.     

Critical evaluation of urinary markers for bladder cancer detection and monitoring

Bladder cancer is currently diagnosed using cystoscopy and cytology in patients with suspicious signs and symptoms. These tests are also used to monitor patients with a history of bladder cancer. The recurrence rate for bladder cancer is high, thus necessitating long-term follow-up. Urine cytology has high specificity but low sensitivity for low-grade bladder tumors. Recently, multiple noninvasive urine-based bladder cancer tests have been developed. Although several markers have been approved by the US Food and Drug Administration for bladder cancer surveillance, only a few are approved for detection of bladder cancer in high-risk patients.     

NMR-based metabolomics study of canine bladder cancer

Bladder cancer is one of the leading lethal cancers worldwide. With the high risk of recurrence for bladder cancer following the initial diagnoses, lifelong monitoring of patients is necessary. The lack of adequate sensitivity and specificity of current noninvasive monitoring approaches including urine cytology, other urine tests, and imaging, underlines the importance of studies that focus on the detection of more reliable biomarkers for this cancer. The emerging area of metabolomics, which deals with the analysis of a large number of small molecules in a single step, promises immense potential for discovering metabolite markers for screening and monitoring treatment response and recurrence in patients with bladder cancer. Since naturally-occurring canine transitional cell carcinoma of the urinary bladder is very similar to human invasive bladder cancer, spontaneous canine transitional cell carcinoma has been applied as a relevant animal model of human invasive transitional cell carcinoma. In this study, we have focused on profiling the metabolites in urine from dogs with transitional cell carcinoma and healthy control dogs combining nuclear magnetic resonance spectroscopy and statistical analysis methods. ¹H NMR-based metabolite profiling analysis was shown to be an effective approach for differentiating samples from dogs with transitional cell carcinoma and healthy controls based on a partial least square-discriminant analysis of the NMR spectra. In addition, there were significant differences in the levels of six individual metabolites between samples from dogs with transitional cell carcinoma and the control group based on the Student's t-test. These metabolites were selected to build a separate partial least square‐discriminant analysis model that was then used to test the classification accuracy. The result showed good classification between transitional cell carcinoma and control groups with the area under the receiver operating characteristic curve of 0.85. The sensitivity and specificity of the model were 86% and 78%, respectively. These results suggest that urine metabolic profiling may have potential for early detection of bladder cancer and of bladder cancer recurrence following treatment, and may enhance our understanding of the mechanisms involved.     

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

Human urine contains a large number of proteins and peptides (the urinary proteome). Global analysis of the human urinary proteome is important for understanding urinary tract diseases. Bladder cancer is the most common urological cancer with higher incidence rates in endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to use the proteomic approach to establish urinary protein biomarkers of bladder cancer. ADAM28, identified by proteomic approaches and confirmed by Western blotting, showed significant differences compared with normal individuals, so it may be a biomarker of bladder cancer.