慢性心力衰竭尿液蛋白标记物的筛选、鉴定及应用研究
摘要
研究背景
慢性心力衰竭(CHF, chronic heart failure)是由于心脏器质性或者功能性疾病损害心脏射血和充盈能力而引起的一组临床综合征,以心功能不全、神经内分泌激活和外周血流分布异常为主要特征。慢性心衰是各种病因导致心血管疾病的严重阶段,其患病率高,死亡率大,5年存活率与恶性肿瘤相仿,严重威胁着患者生命并严重影响患者生活质量,高额的治疗费用给社会经济造成了巨大的负担,成为一个严重的公共卫生问题。缺乏客观诊断标准是心衰死亡率高的原因之一,慢性心力衰竭至今尚无诊断的“金标准”,诊断心衰很大程度取决于医生个人临床判断,主要依据临床症状和体征。目前公认的NYHA分级是最常用的评价心衰严重程度,预测心衰预后的心功能分级系统,但其评价依据是以病人自觉症状为主,其诊断的准确率受到医生进行临床判断的主观影响,同时也与医生的从医经验和患者的配合度相关。目前无创的客观检查手段如超声心动图所能检测的LVEF虽有较大诊断价值,但只能起参考作用。寻找能够反映心衰这一病理生理状态的客观指标是当前面临的主要问题。心衰是一种慢性和发展性疾病,促发心衰的因素有很多,根据神经激素学说及炎性细胞学说,我们找到了以BNP/NT-proBNP,炎症细胞因子(如C反应蛋白,肿瘤坏死因子-α),心肌损伤标记物(如肌钙蛋白)为代表的一系列新的标志物,推动了整体心血管疾病诊治水平的提高；因此有必要在生命活动的直接执行者蛋白质领域上,寻找敏感性高、特异性好的蛋白生物分子标志物。心力衰竭可由多种病理因素引起,在病理情况下蛋白质的变化也是多种多样的。心力衰竭直接相关的分子和细胞改变与临床上心功能受损、疗效、衰竭进程和患者预后的关系并没有确定,不同病因引起的心力衰竭疾病过程,临床表现,预后也有差异,这些差异必然是由其不同的蛋白质特征决定的。直接对心力衰竭过程中蛋白组学的研究是揭示细胞生命本质特征的重要方案,有助于了解心衰时心肌细胞功能障碍及代偿作用的分子机制。本研究采用荧光差异双向凝胶电泳(two-dimensional difference gel electrophoresis,2D-DIGE)蛋白质组学方法结合质谱技术筛选并鉴定慢性心力衰竭和正常人群尿液差异表达蛋白质,并对候选差异表达蛋白质进行验证及进一步的研究。探讨慢性心衰的发生发展机制,寻找慢性心衰特异性分子标记物,为其早期诊断,监测预后及寻求新的治疗靶点提供研究依据。
第一部分慢性心力衰竭患者尿液双向凝胶电泳图谱的构建
1.目的
构建稳定性好,重复率及分辨率高的慢性心力衰竭尿液蛋白双向电泳图谱,为尿液蛋白组水平进一步研究慢性心力衰竭奠定基础。
2.材料和方法
随机选取慢性心力衰竭患者9例,其中心功能分级Ⅱ,Ⅲ和Ⅳ级的心衰患者各3例。标本来源于南方医科大学南方医院心血管内科,明确诊断为慢性心衰的患者。患者纳入标准按照Framingham Heart Study(FHS)的诊断标准诊断为慢性心力衰竭。慢性心衰患者心衰严重程度按照美国纽约心脏学会(NYHA)分级法分级。嘱咐符合纳入标准的患者留取入院后前两天清洁中段晨尿,留取后立即冷却4℃,随后移到-80℃储存。将尿液样本用丙酮或TCA/丙酮沉淀法提取,纯化,浓缩蛋白,并用2D-Quant方法测定蛋白质浓度。随后按照双向电泳手册进行操作。上样样品分为四组：取100μg蛋白样品丙酮处理组(IPG pH3-10NL,24cm),100μg蛋白样品TCA/丙酮处理组(IPG pH3-10NL,24cm),100μg蛋白样品聚焦60KVh组(IPG pH3-10NL,24cm)和100μg蛋白样品聚焦50KVh组(IPG pH3-10NL,24cm).第一向为等电聚焦,聚焦完毕后用平衡液平衡两次,随后胶条移至浓度为10.0%的SDS.PAGE胶上端进行二向电泳,直至溴酚蓝达胶底线。按照固定、敏化、水洗、银染、水洗、显色、终止的步骤对电泳胶染色。用投射扫描仪进行胶图扫描,获取2-DE凝胶图像。
3.结果
1)对比聚焦总伏特数50KVh组；聚焦总伏特数60KVh组的电泳图谱上不仅蛋白点较多,而且横条纹也大大减少,分离效果较好。
2)使用TCA/丙酮沉淀法较丙酮沉淀法的整个图谱效果明显好,背景干净,清晰,图谱的横,竖条纹均较少,且图上的蛋白质点能够清晰分辨,以便进行差异点的辨认和鉴定等后续实验。
4.结论
尿液样本采用TCA/丙酮沉淀法处理,第一向电泳聚焦60kvh的总伏特数能够得到稳定性好,分辨率高、重复性好的双向电泳图谱,为进一步从尿液蛋白组水平筛选出慢性心力衰竭相关蛋白研究奠定了实验基础。
第二部分慢性心力衰竭患者尿液表达变化蛋白质的分离、筛选及鉴定
1.目的
利用2D-DIGE和基质辅助激光解析电离飞行时间质谱鉴定技术对慢性心力衰竭组和正常对照组尿液中的差异蛋白质进行分离,筛选及鉴定,为进一步寻找敏感性高、特异性好的慢性心衰蛋白生物分子标志物奠定基础。
2.材料和方法
本部分研究共纳入正常对照组和慢性心力衰竭组尿液标本各15例,其中心功能分级为Ⅱ,Ⅲ和Ⅳ级的心衰患者各5例。慢性心力衰竭患者尿液标本收集同第一部分。正常对照组标本来源于南方医院体检中心健康受试者的清洁中段晨尿。入选标准为：既往体健,无长期服药史,无重大疾病史的志愿者；无近期感染史；无吸毒及嗜酒史。采用标本混合法,提取两组尿液总蛋白并定量后进行荧光染料标记,Cy2标记内标样品,Cy3标记正常对照组尿液蛋白样品,Cy5标记慢性心力衰竭尿液蛋白样品。待样品标记好后进行双向电泳过程。电泳结束后,标记有荧光染料的凝胶直接用Typhoon9410扫描仪扫描,获取图谱后用Decyder凝胶分析软件进行分析。挑选出28个两组间具有统计学意义(P<0.05)并且差异倍数>1.5倍的显著增加或者显著减少的感兴趣差异蛋白点进行质谱鉴定。利用考马斯亮蓝染色的制备胶上对应的差异点进行质谱鉴定；
3.结果
1)成功利用荧光差异双向电泳方法对慢性心力衰竭和正常对照两组的尿液蛋白进行分离,筛选并获得满意的荧光差异双向电泳图谱；
2)运用Decyder凝胶分析软件对分析后,挑选出慢性心力衰竭和正常对照两组28个差异倍数>1.5倍的差异蛋白点作为质谱鉴定的对象。
3)共鉴定出20种不同的蛋白质。在慢性心力衰竭组中7个升高表达的蛋白分别是：钙粘蛋白-1(Cadherin-1,CDH1),锌α2糖蛋白(Zinc-alpha-2-glycoprotein, AZGP1),α1-酸性糖蛋白(Alpha-1-acid glycoprotein1,ORM1),人α1微球蛋白(Protein AMBP, AMBP),免疫球蛋白K链C区(Ig kappa chain C region,IGKC),人免疫球蛋白轻链kappa(Ig lambda-1chain C regions, IGLC1), GM2神经节苷脂激活因子(Ganglioside GM2activator,GM2A);13个下调表达的蛋白分别是：尿调节素(Uromodulin, UMOD),激肽原-1(Kininogen-1, KNG1),∝l-淀粉酶(Alpha-amylase1, AMY1A),维生素D结合蛋白(Vitamin D-binding protein, GC),胰腺a-淀粉酶(Pancreatic alpha-amylase, AMY2A),血清白蛋白(Serum albumin, ALB),白细胞弹性蛋白酶抑制剂(Leukocyte elastase inhibitor, SERPINB1),间-α-胰蛋白酶抑制剂重链H4(Inter-alpha-trypsininhibitor heavy chain H4,ITIH4),凝血酶原(Prothrombin, F2),钙结合蛋白(Calbindin, CALB1),硫酸乙酰肝素蛋白多糖核心蛋白(Basement membrane-specific heparan sulfate proteoglycan core protein, HSPG2),甘露聚糖结合凝集素丝氨酸蛋白酶2(Mannan-binding lectin serine protease2, MASP2),CD59糖蛋白(CD59glycoprotein,CD59)。
4.结论
顺利利用荧光差异双向电泳对慢性心力衰竭和正常对照两组的尿液蛋白进行分离,筛选及分析差异蛋白点,并成功鉴定出20种不同的蛋白质,7个蛋白质在慢性心力衰竭尿液中高表达,13个低表达。为寻找敏感性高、特异性好的慢性心衰潜在的蛋白生物分子标志物提供了重要的研究基础。
第三部分差异蛋白ORM1在慢性心力衰竭的临床意义研究
1.目的
在鉴定的20个差异蛋白中选取与慢性心力衰竭特异相关的尿液候选蛋白,并研究该候选蛋白与慢性心力衰竭疾病的联系。
2.材料与方法
首选利用生物信息学工具对鉴定的20个差异蛋白进行分析,系统、科学地分析这些鉴定蛋白质累积信息所蕴含的生物学意义。根据生物信息学分析选定ORM1为候选蛋白。利用western blotting技术验证ORM1在尿液中的表达,验证其可靠性后。对62例正常对照组和176例慢性心力衰竭患者的尿液标本用ELISA方法检测ORM1蛋白浓度,其中Ⅱ级患者34例,Ⅲ级患者87例和Ⅳ级心衰患者55例。标本来源于南方医科大学南方医院心血管内科,明确诊断为慢性心衰的患者。患者纳入标准按照Framingham Heart Study(FHS)的诊断标准诊断为慢性心力衰竭。慢性心衰患者严重程度按照美国纽约心脏学会(NYHA)分级法分级。正常对照组标本来源于南方医院体检中心健康受试者的清洁中段晨尿。定量检测后,运用ROC曲线分析尿液ORM1/Cr用来诊断慢性心力衰竭的临界值及对应的特异度和敏感度。此外,根据纳入ELISA研究慢性心力衰竭患者临床资料如：年龄,性别,伴有或无糖尿病,伴有或无高血压,伴有或无冠心病,伴有或无心肌病,伴有或无肾功能损害,eGFR分级,NT-proBNP和和LVEF≥或<50%等进行分析,进一步分析ORM1与慢性心力衰竭之间的关系。
3.结果
1)生物信息学提示这20个差异蛋白共参与了1600个不同的生物学过程,其中前15个生物学过程包括：生长,诱导,信号转导,发病机制,分泌,消化,定位,翻译,凝血,死亡,RNA剪接,细胞凋亡,磷酸化和补体激活等；
2)生物信息学分析及文献挖掘提示ORM1可作为候选蛋白进一步研究；
3)ORM1尿液western blotting结果与蛋白质组学结果一致,在慢性心力衰竭中高表达：
4)慢性心力衰竭组的ORM1/Cr水平为6498.83±4300.21ng/mg,显著高于健康对照组2102.256±1069.24ng/mg。当ORM1/Cr为2484.98ng/mg水平,用其诊断慢性心力衰竭的敏感度为90.91%,特异度为85.48%；
5)慢性心力衰竭的严重程度分组,Ⅱ级,Ⅲ级和Ⅳ级心衰患者尿液中ORM1/Cr水平有显著性差异,浓度依次分别为：3086.24±1474.91ng/mg,6284.97±4088.02ng/mg和8946.71±4298.05ng/mg; Spearman检验提示其相关系数为0.499,尿液中ORM1水平随着心衰程度的加重而升高。
6)慢性心力衰竭患者临床资料的分析中,未发现年龄,性别,伴有或无糖尿病,伴有或无高血压,伴有或无冠心病,伴有或无心肌病,伴有或无肾功能损害,eGFR分级和LVEF>或<50%等分组中尿液ORM1蛋白浓度有显著差异。
4.结论
ORM1蛋白在慢性心力衰竭患者尿液中表达升高,且与心衰严重程度呈正相关关系,尿液ORM1对慢性心力衰竭的诊断及监测有重要的意义,可作为一个尿液蛋白标记物,但其效能和机制需进一步研究。
Backgroud
Chronic heart failure (CHF) is a complex clinical syndrome caused by heart organic or functional disease damages the cardiac ejection and filling capacity, characterized by cardiac dysfunction, neuroendocrine activation and abnormal distribution of peripheral flow. Chronic heart failure is a serious stage for a variety of cardiovascular disease, the morbidity, mortality, and the5-year survival rate of CHF are as high as malignant tumors. CHF serious threat the lives and the quality of life in patients.with high treatment caused tremendous cost to the socio-economic burden has become a serious public health problem. The high cost of chronic heart failure treatment caused tremendous social and economic burden has become a serious public health problem. Lacking of objective diagnostic standard is one reason of the high mortality. There is no Gold Standard in diagnosis of CHF. The diagnosis mainly base on clinical symptoms and signs. Currently,NYHA heart function classification system is most commonly used in evaluating the severity and predicting the prognosis of heart failure, but its evaluation is mainly based on the patient's symptoms.The accuracy of the diagnosis by doctors for clinical judgment not only influenced by subjective factors, but also influenced by doctor's experience and patient's cooperation. The objective non-invasive examination methods, such as LVEF by echocardiography can provide good value on diagnosis, but only to serve as a reference.Therefore searching for an objective index that can reflect physiological pathology of CHF is the main problem we currently facing. CHF is a chronic disease, and it can be caused by many factors, such as inflammatory cells and neural hormone. According to these factors, we found a series of new markers, such as BNP/NT-proBNP, inflammatory cytokine (c-reactive protein, tumor necrosis factor alpha and etc) and markers of myocardial injury (Troponin). These new markers developed the diagnosis and treatment of cardiovascular disease. Therefore it is necessary to find a good protein biomarker, which has the high sensitivity and specificity. Heart failure can be caused by a variety of pathological factors, protein changes also varied in pathological cases. The relationship between molecular and cellular changes in heart failure and impaired cardiac function, the disease process and prognosis of patients has not determined. The process, clinical manifestations, prognosis of heart failure are different, which caused by varieas disease, these differences must be determined by its characteristics of the protein. process Proteomics research on heart failure can reveal the essential characteristics of the cell life programs, help to understand the molecular mechanisms of heart failure, myocardial cell dysfunction and compensatory role.In this study, we used two-dimensional gel electrophoresis (2D-DIGE) approach combined with mass spectrometry(MS) for the identification of urine differentially expressed proteins between chronic heart failure and the normal person, and do more research on candidate differentially expressed proteins. Explore the molecular mechanisms of chronic heart failure, looking for specific biomarkers of chronic heart failure, and provides research basis for its early diagnosis,prognosis, and new therapeutic targets.
Part one:Establishment and further optimization of two-dimensional polyacrylamide gel electrophoresis for proteome analysis of urine sample of chronic heart failure patients
Objective
To establish and optimize two-dimensional electrophoresis (2-DE) of urine sample of chronic heart failure patients aiming to elevate resolution and reproducibility of2-DE map.
Materials and Methods
Random selected nine cases patients with chronic heart failure, including II, Ⅲ and Ⅳ with three cases each. The samples were obtained from Nanfang Hospital Cardiology Department definitive diagnosis for patients with chronic heart failure. The included patients according to the Framingham Heart Study (FHS) diagnostic criteria of chronic heart failure. The severity of chronic heart failure in patients classifyied according to the New York Heart Association (NYHA) classification method. Asked patients who met the inclusion criteria stay the middle of the morning urine specimens after hospitalized two days,then immediately cooled to4℃, and then moved to-80℃storage. Urine samples were extracted with acetone or TCA/acetone precipitation method, purified, concentrated protein, and the protein concentration was determined using2D-Quant. Subsequently, to do next stay in accordance with the two-dimensional electrophoresis manual operation.The sample is divided into four groups:100μg protein sample the acetone treatment group (IPG pH3-10NL,24cm),100μg protein sample TCA/acetone treatment group (IPG pH3-10NL,24cm),100μg protein samples focus60KVh group (IPG pH3-10NL24cm) and100μg protein samples focus50KVh group (IPG pH3-10NL, 24cm).The first step is isoelectric focusing, then balancing twice with a balanced salt solution, followed by strip moved to the upper end of the concentration of10.0%SDS-PAGE gel for two-dimensional polyacrylamide gel electrophoresis until the bromophenol blue reached the bottom line. After fixed, sensitized, washing, silver staining, washing, color, termination, of the steps of gel electrophoresis. UMAXPowerLook1100projection scanner was applied to analyze2DE images.
Results
1. Contrast to the focus total50KVh group; focus the total60KVh group electrophoretic pattern has more protein spots, less horizontal stripes,and also greatly reduced, better separate.
2. TCA/acetone precipitation method is significantly better than acetone precipitation.The entire spectrum has clean background and clear map of the less horizontal, vertical stripes.The proteins on the map are able to clearly distinguish,which are better identified,help for other follow-up experiments.
Conclusion
Urine samples prepared by TCA/acetone precipitation and total of60kvh electrophoresis will be good to get better stability, high resolution, good repeatability dimensional electrophoresis profiles. That is an experimental basis for further perform proteomic research on chronic heart failure from urine samples.
Part two:The Separation, screening and identification of differently expressed proteins in urine samples of Chronic heart failure
Objective
Using2D-DIGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to separate, screen and identify the differentially expressed proteins in the urine from chronic heart failure group and the normal control group, so as to seek high sensitivity and specificity urine biomolecular markers for the chronic heart failure.
Materials and Methods
The part inclued total30cases from the normal control group and chronic heart failure group each15cases of urine specimens, which Ⅱ, Ⅲ and Ⅳ level five cases each. Patients with chronic heart failure urine specimens collected as the first part. The normal control group specimens from the clean morning urine of the Southern Hospital Medical Center in healthy subjects. Inclusion criteria were: previously healthy volunteers without long-term medication history, no history of major diseases; history of recent infection; no history of drug addicts and alcoholics.Using mixed specimens method, to extract two sets of urine total protein and quantitative fluorescent dye marker, Cy2labeled internal standard samples, Cy3-labeled normal control group urine protein sample, Cy5marked chronic heart failure urine protein samples. Two-dimensional electrophoresis process can be used after samples marked. After electrophoresis, the gel labeled with fluorescent dye directly scaned with Typhoon9410scanner scans to get picture,and then Decyder gel analysis software for analysis. Selected28between the two groups was statistically significant (P<0.05) and the difference in multiples of>1.5-fold increase, or are interested in significantly reducing the differential protein spots were identified by mass spectrometry. staining preparation of gum on the corresponding point of difference identified; After staining by using Coomassie blue, the corresponding different protein spots form the preparing gel was identified by mass spectrometry.
Results
1. Successful used of two-dimensional difference gel electrophoresis method separate urine protein between chronic heart failure patients and normal control groups.We first time established2D-DIGE gel protein images of CHF and identified screen different protein spots.
2. After using Decyder gel analysis software, selected28different protein spots which differences in multiples of>1.5-fold between chronic heart failure and normal control were identified by mass spectrometry.
3. Identified a total of20different proteins,7up-regulated in chronic heart failure were:Cadherin-1(CDH1),Zinc-alpha-2-glycoprotein(AZGP1), Alpha-1-acid glycoprotein1(ORM1),Protein AMBP(AMBP),Ig kappa chain C region(IGKC), Ig lambda-1chain C regions(IGLC1), Ganglioside GM2activator(GM2A);13down-regulated proteins were:Uromodulin,(UMOD), Kininogen-1(KNG1), Alpha-amylase1(AMY1A), Vitamin D-binding protein(GC), Pancreatic alpha-amylase(AMY2A),Serum albumin(ALB), Leukocyte elastase inhibitor(SERPINB1), Inter-alpha-trypsin inhibitor heavy chain H4(ITIH4), Prothrombin(F2), Calbindin(CALB1), Basement membrane-specific heparan sulfate proteoglycan core protein(HSPG2), Mannan-binding lectin serine protease2(MASP2),CD59glycoprotein(CD59).
Conclusion
Successful used of two-dimensional difference gel electrophoresis method separate urine protein between chronic heart failure patients and normal control groups. We first time established2D-DIGE gel protein images of CHF and identified screen different protein spots. Identified a total of20different proteins,7up-regulated and13down-regulated in chronic heart failure. That is an experimental basis for further look for high sensitivity and specificity potential urinary protein biomolecular on chronic heart failure.
Part three:The clinical significance Of differential proteins ORM1on Chronic heart failure
Objective
To select candidate protein which has some specific significance on chronic heart failure in20differentially expressed proteins, and to research the relationship between candidate protein and chronic heart failure.
Materials and Methods
First, using bioinformatics tools to analysis identified20differentially expressed proteins, statistical and scientific analysis of the information contained in these identified proteins which accumulated some biological significance. According to bioinformatics analysis, we selected ORM1as candidate proteins. Using western blotting to confirm its reliability of ORM1in urine expression.
Using ELISA to detect ORM1protein concentration on urine samples of62control group and176patients with chronic heart failure. Of those176patients NYHA stage were:34cases of patients with stage Ⅱ,87cases with stage III and55cases with stage Ⅳ. The samples were obtained from Nanfang Hospital Cardiology Department definitive diagnosis for patients with chronic heart failure. The included patients according to the Framingham Heart Study (FHS) diagnostic criteria of chronic heart failure. The severity of chronic heart failure in patients classifyied according to the New York Heart Association (NYHA) classification method. The normal control group specimens from the clean morning urine of the Southern Hospital Medical Center in healthy subjects. After quantitative detection,using ROC curve analysis the urine ORM1/Cr to diagnose chronic heart failure threshold and the corresponding specificity and sensitivity. In addition, according to clinical data in patients with chronic heart failure such as:age, gender, diabetes mellitus, eGFR,NT-proBNP,with or without hypertension, coronary heart disease, renal dysfunction and LVEF≥or<50%, do more subgroup analysis, further analysis the relationship between ORM1and chronic heart failure.
Results
1. Bioinformatics prompted20differentially expressed proteins were involved in 1600in different biological processes, the top15biological processes including: growth, induction, pathogenesis, secretion, digestion, positioning, translation, coagulation, death, RNA splicing, apoptosis, and complement activation;
2. According to bioinformatics analysis and literature review,ORM1can further study as a candidate protein;
3. Western blotting of urinary ORM1to verify the results2D-DIGE,consist high expression in patients with chronic heart failure;
4. The ORM1/Cr level of chronic heart failure group was6498.83±4300.21ng/mg, significantly higher than the healthy control group2102.256±1069.24ng/mg. ROC analyses rendered a cut-off value with1407.481ng/mg corresponding to90.91%sensitivity and85.48%specificity d to diagnosis of chronic heart failure;
5. There were significantly different ORM1levels in chronic heart failure patients. The concentrations of ORM1in Ⅱ,ⅢandⅣ patients were3086.24±1474.91ng/mg,6284.97±4088.02ng/mg and8946.71±4298.05ng/mg respectively. The correlation indicated by Spearman test is0.499. The urine ORM1level rises as heart failure severity increasing.
6. Among the subgroup analyses on the basis of the clinical data of patients with chronic heart failure, the urine ORM1protein concentrations were no significant differences on age, sex, diabetes, hypertension, coronary heart disease, cardiomyopathy, renal dysfunction,eGFR,and LVEF.
Conclusion
The ORM1is elevated in the urine of patients with chronic heart failure, and is positively associated with the severity of heart failure. ORM1has an important significance on diagnosis and monitoring of chronic heart failure. It could be a urinary marker, but its effectiveness and mechanisms need to be studied in the future.
慢性心力衰竭(CHF, chronic heart failure)是由于心脏器质性或者功能性疾病损害心脏射血和充盈能力而引起的一组临床综合征,以心功能不全、神经内分泌激活和外周血流分布异常为主要特征。慢性心衰是各种病因导致心血管疾病的严重阶段,其患病率高,死亡率大,5年存活率与恶性肿瘤相仿,严重威胁着患者生命并严重影响患者生活质量,高额的治疗费用给社会经济造成了巨大的负担,成为一个严重的公共卫生问题。缺乏客观诊断标准是心衰死亡率高的原因之一,慢性心力衰竭至今尚无诊断的“金标准”,诊断心衰很大程度取决于医生个人临床判断,主要依据临床症状和体征。目前公认的NYHA分级是最常用的评价心衰严重程度,预测心衰预后的心功能分级系统,但其评价依据是以病人自觉症状为主,其诊断的准确率受到医生进行临床判断的主观影响,同时也与医生的从医经验和患者的配合度相关。目前无创的客观检查手段如超声心动图所能检测的LVEF虽有较大诊断价值,但只能起参考作用。寻找能够反映心衰这一病理生理状态的客观指标是当前面临的主要问题。心衰是一种慢性和发展性疾病,促发心衰的因素有很多,根据神经激素学说及炎性细胞学说,我们找到了以BNP/NT-proBNP,炎症细胞因子(如C反应蛋白,肿瘤坏死因子-α),心肌损伤标记物(如肌钙蛋白)为代表的一系列新的标志物,推动了整体心血管疾病诊治水平的提高；因此有必要在生命活动的直接执行者蛋白质领域上,寻找敏感性高、特异性好的蛋白生物分子标志物。心力衰竭可由多种病理因素引起,在病理情况下蛋白质的变化也是多种多样的。心力衰竭直接相关的分子和细胞改变与临床上心功能受损、疗效、衰竭进程和患者预后的关系并没有确定,不同病因引起的心力衰竭疾病过程,临床表现,预后也有差异,这些差异必然是由其不同的蛋白质特征决定的。直接对心力衰竭过程中蛋白组学的研究是揭示细胞生命本质特征的重要方案,有助于了解心衰时心肌细胞功能障碍及代偿作用的分子机制。本研究采用荧光差异双向凝胶电泳(two-dimensional difference gel electrophoresis,2D-DIGE)蛋白质组学方法结合质谱技术筛选并鉴定慢性心力衰竭和正常人群尿液差异表达蛋白质,并对候选差异表达蛋白质进行验证及进一步的研究。探讨慢性心衰的发生发展机制,寻找慢性心衰特异性分子标记物,为其早期诊断,监测预后及寻求新的治疗靶点提供研究依据。
第一部分慢性心力衰竭患者尿液双向凝胶电泳图谱的构建
1.目的
构建稳定性好,重复率及分辨率高的慢性心力衰竭尿液蛋白双向电泳图谱,为尿液蛋白组水平进一步研究慢性心力衰竭奠定基础。
2.材料和方法
随机选取慢性心力衰竭患者9例,其中心功能分级Ⅱ,Ⅲ和Ⅳ级的心衰患者各3例。标本来源于南方医科大学南方医院心血管内科,明确诊断为慢性心衰的患者。患者纳入标准按照Framingham Heart Study(FHS)的诊断标准诊断为慢性心力衰竭。慢性心衰患者心衰严重程度按照美国纽约心脏学会(NYHA)分级法分级。嘱咐符合纳入标准的患者留取入院后前两天清洁中段晨尿,留取后立即冷却4℃,随后移到-80℃储存。将尿液样本用丙酮或TCA/丙酮沉淀法提取,纯化,浓缩蛋白,并用2D-Quant方法测定蛋白质浓度。随后按照双向电泳手册进行操作。上样样品分为四组：取100μg蛋白样品丙酮处理组(IPG pH3-10NL,24cm),100μg蛋白样品TCA/丙酮处理组(IPG pH3-10NL,24cm),100μg蛋白样品聚焦60KVh组(IPG pH3-10NL,24cm)和100μg蛋白样品聚焦50KVh组(IPG pH3-10NL,24cm).第一向为等电聚焦,聚焦完毕后用平衡液平衡两次,随后胶条移至浓度为10.0%的SDS.PAGE胶上端进行二向电泳,直至溴酚蓝达胶底线。按照固定、敏化、水洗、银染、水洗、显色、终止的步骤对电泳胶染色。用投射扫描仪进行胶图扫描,获取2-DE凝胶图像。
3.结果
1)对比聚焦总伏特数50KVh组；聚焦总伏特数60KVh组的电泳图谱上不仅蛋白点较多,而且横条纹也大大减少,分离效果较好。
2)使用TCA/丙酮沉淀法较丙酮沉淀法的整个图谱效果明显好,背景干净,清晰,图谱的横,竖条纹均较少,且图上的蛋白质点能够清晰分辨,以便进行差异点的辨认和鉴定等后续实验。
4.结论
尿液样本采用TCA/丙酮沉淀法处理,第一向电泳聚焦60kvh的总伏特数能够得到稳定性好,分辨率高、重复性好的双向电泳图谱,为进一步从尿液蛋白组水平筛选出慢性心力衰竭相关蛋白研究奠定了实验基础。
第二部分慢性心力衰竭患者尿液表达变化蛋白质的分离、筛选及鉴定
1.目的
利用2D-DIGE和基质辅助激光解析电离飞行时间质谱鉴定技术对慢性心力衰竭组和正常对照组尿液中的差异蛋白质进行分离,筛选及鉴定,为进一步寻找敏感性高、特异性好的慢性心衰蛋白生物分子标志物奠定基础。
2.材料和方法
本部分研究共纳入正常对照组和慢性心力衰竭组尿液标本各15例,其中心功能分级为Ⅱ,Ⅲ和Ⅳ级的心衰患者各5例。慢性心力衰竭患者尿液标本收集同第一部分。正常对照组标本来源于南方医院体检中心健康受试者的清洁中段晨尿。入选标准为：既往体健,无长期服药史,无重大疾病史的志愿者；无近期感染史；无吸毒及嗜酒史。采用标本混合法,提取两组尿液总蛋白并定量后进行荧光染料标记,Cy2标记内标样品,Cy3标记正常对照组尿液蛋白样品,Cy5标记慢性心力衰竭尿液蛋白样品。待样品标记好后进行双向电泳过程。电泳结束后,标记有荧光染料的凝胶直接用Typhoon9410扫描仪扫描,获取图谱后用Decyder凝胶分析软件进行分析。挑选出28个两组间具有统计学意义(P<0.05)并且差异倍数>1.5倍的显著增加或者显著减少的感兴趣差异蛋白点进行质谱鉴定。利用考马斯亮蓝染色的制备胶上对应的差异点进行质谱鉴定；
3.结果
1)成功利用荧光差异双向电泳方法对慢性心力衰竭和正常对照两组的尿液蛋白进行分离,筛选并获得满意的荧光差异双向电泳图谱；
2)运用Decyder凝胶分析软件对分析后,挑选出慢性心力衰竭和正常对照两组28个差异倍数>1.5倍的差异蛋白点作为质谱鉴定的对象。
3)共鉴定出20种不同的蛋白质。在慢性心力衰竭组中7个升高表达的蛋白分别是：钙粘蛋白-1(Cadherin-1,CDH1),锌α2糖蛋白(Zinc-alpha-2-glycoprotein, AZGP1),α1-酸性糖蛋白(Alpha-1-acid glycoprotein1,ORM1),人α1微球蛋白(Protein AMBP, AMBP),免疫球蛋白K链C区(Ig kappa chain C region,IGKC),人免疫球蛋白轻链kappa(Ig lambda-1chain C regions, IGLC1), GM2神经节苷脂激活因子(Ganglioside GM2activator,GM2A);13个下调表达的蛋白分别是：尿调节素(Uromodulin, UMOD),激肽原-1(Kininogen-1, KNG1),∝l-淀粉酶(Alpha-amylase1, AMY1A),维生素D结合蛋白(Vitamin D-binding protein, GC),胰腺a-淀粉酶(Pancreatic alpha-amylase, AMY2A),血清白蛋白(Serum albumin, ALB),白细胞弹性蛋白酶抑制剂(Leukocyte elastase inhibitor, SERPINB1),间-α-胰蛋白酶抑制剂重链H4(Inter-alpha-trypsininhibitor heavy chain H4,ITIH4),凝血酶原(Prothrombin, F2),钙结合蛋白(Calbindin, CALB1),硫酸乙酰肝素蛋白多糖核心蛋白(Basement membrane-specific heparan sulfate proteoglycan core protein, HSPG2),甘露聚糖结合凝集素丝氨酸蛋白酶2(Mannan-binding lectin serine protease2, MASP2),CD59糖蛋白(CD59glycoprotein,CD59)。
4.结论
顺利利用荧光差异双向电泳对慢性心力衰竭和正常对照两组的尿液蛋白进行分离,筛选及分析差异蛋白点,并成功鉴定出20种不同的蛋白质,7个蛋白质在慢性心力衰竭尿液中高表达,13个低表达。为寻找敏感性高、特异性好的慢性心衰潜在的蛋白生物分子标志物提供了重要的研究基础。
第三部分差异蛋白ORM1在慢性心力衰竭的临床意义研究
1.目的
在鉴定的20个差异蛋白中选取与慢性心力衰竭特异相关的尿液候选蛋白,并研究该候选蛋白与慢性心力衰竭疾病的联系。
2.材料与方法
首选利用生物信息学工具对鉴定的20个差异蛋白进行分析,系统、科学地分析这些鉴定蛋白质累积信息所蕴含的生物学意义。根据生物信息学分析选定ORM1为候选蛋白。利用western blotting技术验证ORM1在尿液中的表达,验证其可靠性后。对62例正常对照组和176例慢性心力衰竭患者的尿液标本用ELISA方法检测ORM1蛋白浓度,其中Ⅱ级患者34例,Ⅲ级患者87例和Ⅳ级心衰患者55例。标本来源于南方医科大学南方医院心血管内科,明确诊断为慢性心衰的患者。患者纳入标准按照Framingham Heart Study(FHS)的诊断标准诊断为慢性心力衰竭。慢性心衰患者严重程度按照美国纽约心脏学会(NYHA)分级法分级。正常对照组标本来源于南方医院体检中心健康受试者的清洁中段晨尿。定量检测后,运用ROC曲线分析尿液ORM1/Cr用来诊断慢性心力衰竭的临界值及对应的特异度和敏感度。此外,根据纳入ELISA研究慢性心力衰竭患者临床资料如：年龄,性别,伴有或无糖尿病,伴有或无高血压,伴有或无冠心病,伴有或无心肌病,伴有或无肾功能损害,eGFR分级,NT-proBNP和和LVEF≥或<50%等进行分析,进一步分析ORM1与慢性心力衰竭之间的关系。
3.结果
1)生物信息学提示这20个差异蛋白共参与了1600个不同的生物学过程,其中前15个生物学过程包括：生长,诱导,信号转导,发病机制,分泌,消化,定位,翻译,凝血,死亡,RNA剪接,细胞凋亡,磷酸化和补体激活等；
2)生物信息学分析及文献挖掘提示ORM1可作为候选蛋白进一步研究；
3)ORM1尿液western blotting结果与蛋白质组学结果一致,在慢性心力衰竭中高表达：
4)慢性心力衰竭组的ORM1/Cr水平为6498.83±4300.21ng/mg,显著高于健康对照组2102.256±1069.24ng/mg。当ORM1/Cr为2484.98ng/mg水平,用其诊断慢性心力衰竭的敏感度为90.91%,特异度为85.48%；
5)慢性心力衰竭的严重程度分组,Ⅱ级,Ⅲ级和Ⅳ级心衰患者尿液中ORM1/Cr水平有显著性差异,浓度依次分别为：3086.24±1474.91ng/mg,6284.97±4088.02ng/mg和8946.71±4298.05ng/mg; Spearman检验提示其相关系数为0.499,尿液中ORM1水平随着心衰程度的加重而升高。
6)慢性心力衰竭患者临床资料的分析中,未发现年龄,性别,伴有或无糖尿病,伴有或无高血压,伴有或无冠心病,伴有或无心肌病,伴有或无肾功能损害,eGFR分级和LVEF>或<50%等分组中尿液ORM1蛋白浓度有显著差异。
4.结论
ORM1蛋白在慢性心力衰竭患者尿液中表达升高,且与心衰严重程度呈正相关关系,尿液ORM1对慢性心力衰竭的诊断及监测有重要的意义,可作为一个尿液蛋白标记物,但其效能和机制需进一步研究。
Backgroud
Chronic heart failure (CHF) is a complex clinical syndrome caused by heart organic or functional disease damages the cardiac ejection and filling capacity, characterized by cardiac dysfunction, neuroendocrine activation and abnormal distribution of peripheral flow. Chronic heart failure is a serious stage for a variety of cardiovascular disease, the morbidity, mortality, and the5-year survival rate of CHF are as high as malignant tumors. CHF serious threat the lives and the quality of life in patients.with high treatment caused tremendous cost to the socio-economic burden has become a serious public health problem. The high cost of chronic heart failure treatment caused tremendous social and economic burden has become a serious public health problem. Lacking of objective diagnostic standard is one reason of the high mortality. There is no Gold Standard in diagnosis of CHF. The diagnosis mainly base on clinical symptoms and signs. Currently,NYHA heart function classification system is most commonly used in evaluating the severity and predicting the prognosis of heart failure, but its evaluation is mainly based on the patient's symptoms.The accuracy of the diagnosis by doctors for clinical judgment not only influenced by subjective factors, but also influenced by doctor's experience and patient's cooperation. The objective non-invasive examination methods, such as LVEF by echocardiography can provide good value on diagnosis, but only to serve as a reference.Therefore searching for an objective index that can reflect physiological pathology of CHF is the main problem we currently facing. CHF is a chronic disease, and it can be caused by many factors, such as inflammatory cells and neural hormone. According to these factors, we found a series of new markers, such as BNP/NT-proBNP, inflammatory cytokine (c-reactive protein, tumor necrosis factor alpha and etc) and markers of myocardial injury (Troponin). These new markers developed the diagnosis and treatment of cardiovascular disease. Therefore it is necessary to find a good protein biomarker, which has the high sensitivity and specificity. Heart failure can be caused by a variety of pathological factors, protein changes also varied in pathological cases. The relationship between molecular and cellular changes in heart failure and impaired cardiac function, the disease process and prognosis of patients has not determined. The process, clinical manifestations, prognosis of heart failure are different, which caused by varieas disease, these differences must be determined by its characteristics of the protein. process Proteomics research on heart failure can reveal the essential characteristics of the cell life programs, help to understand the molecular mechanisms of heart failure, myocardial cell dysfunction and compensatory role.In this study, we used two-dimensional gel electrophoresis (2D-DIGE) approach combined with mass spectrometry(MS) for the identification of urine differentially expressed proteins between chronic heart failure and the normal person, and do more research on candidate differentially expressed proteins. Explore the molecular mechanisms of chronic heart failure, looking for specific biomarkers of chronic heart failure, and provides research basis for its early diagnosis,prognosis, and new therapeutic targets.
Part one:Establishment and further optimization of two-dimensional polyacrylamide gel electrophoresis for proteome analysis of urine sample of chronic heart failure patients
Objective
To establish and optimize two-dimensional electrophoresis (2-DE) of urine sample of chronic heart failure patients aiming to elevate resolution and reproducibility of2-DE map.
Materials and Methods
Random selected nine cases patients with chronic heart failure, including II, Ⅲ and Ⅳ with three cases each. The samples were obtained from Nanfang Hospital Cardiology Department definitive diagnosis for patients with chronic heart failure. The included patients according to the Framingham Heart Study (FHS) diagnostic criteria of chronic heart failure. The severity of chronic heart failure in patients classifyied according to the New York Heart Association (NYHA) classification method. Asked patients who met the inclusion criteria stay the middle of the morning urine specimens after hospitalized two days,then immediately cooled to4℃, and then moved to-80℃storage. Urine samples were extracted with acetone or TCA/acetone precipitation method, purified, concentrated protein, and the protein concentration was determined using2D-Quant. Subsequently, to do next stay in accordance with the two-dimensional electrophoresis manual operation.The sample is divided into four groups:100μg protein sample the acetone treatment group (IPG pH3-10NL,24cm),100μg protein sample TCA/acetone treatment group (IPG pH3-10NL,24cm),100μg protein samples focus60KVh group (IPG pH3-10NL24cm) and100μg protein samples focus50KVh group (IPG pH3-10NL, 24cm).The first step is isoelectric focusing, then balancing twice with a balanced salt solution, followed by strip moved to the upper end of the concentration of10.0%SDS-PAGE gel for two-dimensional polyacrylamide gel electrophoresis until the bromophenol blue reached the bottom line. After fixed, sensitized, washing, silver staining, washing, color, termination, of the steps of gel electrophoresis. UMAXPowerLook1100projection scanner was applied to analyze2DE images.
Results
1. Contrast to the focus total50KVh group; focus the total60KVh group electrophoretic pattern has more protein spots, less horizontal stripes,and also greatly reduced, better separate.
2. TCA/acetone precipitation method is significantly better than acetone precipitation.The entire spectrum has clean background and clear map of the less horizontal, vertical stripes.The proteins on the map are able to clearly distinguish,which are better identified,help for other follow-up experiments.
Conclusion
Urine samples prepared by TCA/acetone precipitation and total of60kvh electrophoresis will be good to get better stability, high resolution, good repeatability dimensional electrophoresis profiles. That is an experimental basis for further perform proteomic research on chronic heart failure from urine samples.
Part two:The Separation, screening and identification of differently expressed proteins in urine samples of Chronic heart failure
Objective
Using2D-DIGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to separate, screen and identify the differentially expressed proteins in the urine from chronic heart failure group and the normal control group, so as to seek high sensitivity and specificity urine biomolecular markers for the chronic heart failure.
Materials and Methods
The part inclued total30cases from the normal control group and chronic heart failure group each15cases of urine specimens, which Ⅱ, Ⅲ and Ⅳ level five cases each. Patients with chronic heart failure urine specimens collected as the first part. The normal control group specimens from the clean morning urine of the Southern Hospital Medical Center in healthy subjects. Inclusion criteria were: previously healthy volunteers without long-term medication history, no history of major diseases; history of recent infection; no history of drug addicts and alcoholics.Using mixed specimens method, to extract two sets of urine total protein and quantitative fluorescent dye marker, Cy2labeled internal standard samples, Cy3-labeled normal control group urine protein sample, Cy5marked chronic heart failure urine protein samples. Two-dimensional electrophoresis process can be used after samples marked. After electrophoresis, the gel labeled with fluorescent dye directly scaned with Typhoon9410scanner scans to get picture,and then Decyder gel analysis software for analysis. Selected28between the two groups was statistically significant (P<0.05) and the difference in multiples of>1.5-fold increase, or are interested in significantly reducing the differential protein spots were identified by mass spectrometry. staining preparation of gum on the corresponding point of difference identified; After staining by using Coomassie blue, the corresponding different protein spots form the preparing gel was identified by mass spectrometry.
Results
1. Successful used of two-dimensional difference gel electrophoresis method separate urine protein between chronic heart failure patients and normal control groups.We first time established2D-DIGE gel protein images of CHF and identified screen different protein spots.
2. After using Decyder gel analysis software, selected28different protein spots which differences in multiples of>1.5-fold between chronic heart failure and normal control were identified by mass spectrometry.
3. Identified a total of20different proteins,7up-regulated in chronic heart failure were:Cadherin-1(CDH1),Zinc-alpha-2-glycoprotein(AZGP1), Alpha-1-acid glycoprotein1(ORM1),Protein AMBP(AMBP),Ig kappa chain C region(IGKC), Ig lambda-1chain C regions(IGLC1), Ganglioside GM2activator(GM2A);13down-regulated proteins were:Uromodulin,(UMOD), Kininogen-1(KNG1), Alpha-amylase1(AMY1A), Vitamin D-binding protein(GC), Pancreatic alpha-amylase(AMY2A),Serum albumin(ALB), Leukocyte elastase inhibitor(SERPINB1), Inter-alpha-trypsin inhibitor heavy chain H4(ITIH4), Prothrombin(F2), Calbindin(CALB1), Basement membrane-specific heparan sulfate proteoglycan core protein(HSPG2), Mannan-binding lectin serine protease2(MASP2),CD59glycoprotein(CD59).
Conclusion
Successful used of two-dimensional difference gel electrophoresis method separate urine protein between chronic heart failure patients and normal control groups. We first time established2D-DIGE gel protein images of CHF and identified screen different protein spots. Identified a total of20different proteins,7up-regulated and13down-regulated in chronic heart failure. That is an experimental basis for further look for high sensitivity and specificity potential urinary protein biomolecular on chronic heart failure.
Part three:The clinical significance Of differential proteins ORM1on Chronic heart failure
Objective
To select candidate protein which has some specific significance on chronic heart failure in20differentially expressed proteins, and to research the relationship between candidate protein and chronic heart failure.
Materials and Methods
First, using bioinformatics tools to analysis identified20differentially expressed proteins, statistical and scientific analysis of the information contained in these identified proteins which accumulated some biological significance. According to bioinformatics analysis, we selected ORM1as candidate proteins. Using western blotting to confirm its reliability of ORM1in urine expression.
Using ELISA to detect ORM1protein concentration on urine samples of62control group and176patients with chronic heart failure. Of those176patients NYHA stage were:34cases of patients with stage Ⅱ,87cases with stage III and55cases with stage Ⅳ. The samples were obtained from Nanfang Hospital Cardiology Department definitive diagnosis for patients with chronic heart failure. The included patients according to the Framingham Heart Study (FHS) diagnostic criteria of chronic heart failure. The severity of chronic heart failure in patients classifyied according to the New York Heart Association (NYHA) classification method. The normal control group specimens from the clean morning urine of the Southern Hospital Medical Center in healthy subjects. After quantitative detection,using ROC curve analysis the urine ORM1/Cr to diagnose chronic heart failure threshold and the corresponding specificity and sensitivity. In addition, according to clinical data in patients with chronic heart failure such as:age, gender, diabetes mellitus, eGFR,NT-proBNP,with or without hypertension, coronary heart disease, renal dysfunction and LVEF≥or<50%, do more subgroup analysis, further analysis the relationship between ORM1and chronic heart failure.
Results
1. Bioinformatics prompted20differentially expressed proteins were involved in 1600in different biological processes, the top15biological processes including: growth, induction, pathogenesis, secretion, digestion, positioning, translation, coagulation, death, RNA splicing, apoptosis, and complement activation;
2. According to bioinformatics analysis and literature review,ORM1can further study as a candidate protein;
3. Western blotting of urinary ORM1to verify the results2D-DIGE,consist high expression in patients with chronic heart failure;
4. The ORM1/Cr level of chronic heart failure group was6498.83±4300.21ng/mg, significantly higher than the healthy control group2102.256±1069.24ng/mg. ROC analyses rendered a cut-off value with1407.481ng/mg corresponding to90.91%sensitivity and85.48%specificity d to diagnosis of chronic heart failure;
5. There were significantly different ORM1levels in chronic heart failure patients. The concentrations of ORM1in Ⅱ,ⅢandⅣ patients were3086.24±1474.91ng/mg,6284.97±4088.02ng/mg and8946.71±4298.05ng/mg respectively. The correlation indicated by Spearman test is0.499. The urine ORM1level rises as heart failure severity increasing.
6. Among the subgroup analyses on the basis of the clinical data of patients with chronic heart failure, the urine ORM1protein concentrations were no significant differences on age, sex, diabetes, hypertension, coronary heart disease, cardiomyopathy, renal dysfunction,eGFR,and LVEF.
Conclusion
The ORM1is elevated in the urine of patients with chronic heart failure, and is positively associated with the severity of heart failure. ORM1has an important significance on diagnosis and monitoring of chronic heart failure. It could be a urinary marker, but its effectiveness and mechanisms need to be studied in the future.
引文
[1]中国心血管健康多中心合作研究组.中国心力衰竭流行病学调查及其患病率.中华心血管病杂志,2003,31(1)：3-6.
[2]Gheorghiade M, Bonow RO. Chronic heart failure in the United States:am an ifestation of coronary artery disease.Circulation,1998,97(3):282-289.
[3]中华医学会心血管病学分会.中国部分地区1980、1990、2000年慢性心力衰竭住院病例回顾性调查.中华心血管病杂志,2002,30(8)：450-454.
[4]Lin D, Hollander Z, Meredith A, et al.Molecular signatures of end-stage heart failure.J Card Fail.2011;17:867-74.
[5]Hunt SA, Abraham WT, Chin MH, et al. ACC/AHA 2005 guideline update for the diagnosis and management of chronic heart failure in the adult. Circulation,2005,112:1825-1852.
[6]ACC/AHA. Guidelines for the evaluation and management of chronic heart failure. Circulation,2001,104:2996-3007.
[7]顾东风,黄广勇,何江.中国心力衰竭流行病学调查及其患病率.中华心血管病杂志,2003,31(1)：03-06.
[8]AHA 2002 heart and stroke statistical update[M]. Dallas:American Heart Association,2001.1.
[9]Panagopoulou V, Deftereos S, Kossyvakis C, et al.NTproBNP:An Important Biomarker in Cardiac Diseases. Curr Top Med Chem.2013 Mar 4.
[10]Cohen-Solal.Guidelines for the diagnosis and treatment of chronic heart failure: task force for the diagnosis and treatment of chronic heart failure. Eur Heart J, 2001,22:1527-1560.
[11]McMurray JJ, Adamopoulos S, Anker SD, et al.ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012:The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC.Eur J Heart Fail.2012 Aug; 14(8):803-69.
[12]Cadete VJ, Lin HB, Sawicka J, et al.Proteomic analysis of right and left cardiac ventricles under aerobic conditions and after ischemia/reperfusion.Proteomics. 2012 Jun 11.
[13]Haas B, Serchi T, Wagner DR,et al. Proteomic analysis of plasma samples from patients with acute myocardial infarction identifies haptoglobin as a potential prognostic biomarker.Proteomics.2011 Dec 10;75(1):229-36.
[14]Wilkins MR,Williams KL,Hohstrasser DF,et al. Proteome Research:New frontiers in fuctional genomics. [M].New York:Springer,1997.
[15]Maggio ET, Ramnarayan K.Recent developments in computational proteomics. Drug Discov Today.2001 Oct 1;6(19):996-1004.
[16]O'Farrell PH.High resolution two-dimensional electrophoresis of proteins. J Biol Chem.1975 May 25;250(10):4007-21.
[17]Zhang J, Song MQ, Zhu JS,et,al.Identification of differentially-expressed proteins between early submucosal non-invasive and invasive colorectal cancer using 2D-DIGE and mass spectrometry. Int J Immunopathol Pharmacol.2011 Oct-Dec;24(4):849-59.
[18]Bigot P, Mouzat K, Lebdai S,et al. Quantitative proteomic determination of diethylstilbestrol action on prostate cancer. Asian J Androl.2013 May; 15(3):413-20.
[19]Wu WW, Wang G, Baek SJ, Shen RF.Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF. J Proteome Res.2006 Mar;5(3):651-8.
[20]成海平,钱小红.蛋白质组研究的技术体系及其进展.[J].生物化学与生物物理进展,2000,27(6)：664-667.
[21]Wright ME, Han DK, Aebersold R. Mass spectrometry-based expression profiling of clinical prostate cancer. Mol Cell Proteomics.2005 Apr;4(4):545-54.
[22]Camacho-Carvajal MM, Wollscheid B, Aebersold R,Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates:a proteomics approach. Mol Cell Proteomics.2004 Feb;3(2):176-82.
[23]Patton WF.Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci.2002 May 5;771(1-2):3-31.
[24]Hamdan M, Righetti PG. Modern strategies for protein quantification in proteome analysis:advantages and limitations. Mass Spectrom Rev.2002 Jul-Aug;21(4):287-302.
[25]Donahue MP, Rose K, Hochstrasser D,et al.Discovery of proteins related to coronary artery disease using industrial-scale proteomics analysis of pooled plasma. Am Heart J.2006 Sep;152(3):478-85.
[26]李新华,张万岱,肖冰等.蛋白质组学及其在人类疾病中的应用.中华内科杂志,2003,42(2)：133-134.
[27]张伟英,赵猛,兰小鹏.蛋白质组学技术在心血管疾病研究中的应用.心血管康复医学杂志,2009,8(18)：403-405.
[28]Weekes J, Wheeler CH, Yan JX, et, al.Bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy. Electrophoresis.1999 Apr-May;20(4-5):898-906.
[29]De Celle T, Vanrobaeys F, Lijnen P, et al.Alterations in mouse cardiac proteome after in vivo myocardial infarction:permanent ischaemia versus ischaemia-reperfusion. Exp Physiol.2005 Jul;90(4):593-606.
[30]Jungblut PR, Zimny-Arndt U, Zeindl-Eberhart E et al.Proteomics in human disease:cancer, heart and infectious diseases.Electrophoresis.1999 Jul;20(10):2100-10.
[31]段海峰,钱令嘉,应万涛,等.应激心肌细胞蛋白质组双向凝胶电泳分析.中国生物化学分子生物学报,2002,18(2)：234-239.
[32]Jin X, X ia L, W ang LS, et a.l Differential protein expression in hypertrophic heart with and without hypertension in spontaneously hypertensive rats. Proteom ics,2006,6 (6):1948-1956.
[33]Abdul S alam VB, Pau 1 GA, A 1 i JO, et al.Identification of plasm a protein biomarkers associated with idiopathic pulmonary arterial hypertension. Proteomics,2006,6 (7):2286-2294.
[34]Zimmerli LU, Schiffer E, Zurbig P, et al.Urinary proteomic biomarkers in coronary artery disease. Mol Cell Proteomics.2008 Feb;7(2):290-8.
[35]Kuznetsova T, Mischak H, Mullen W, Staessen JA. Urinary proteome analysis in hypertensive patients with left ventricular diastolic dysfunction. Eur Heart J. 2012 Sep;33(18):2342-50.
[36]Heinke MY, Wheeler CH, Yan JX, et al.Changes in myocardial protein expression in pacing-induced canine heart failure. Electrophoresis.1999 Jul;20(10):2086-93.
[37]Jiang L, Tsubakihara M, Heinke MY,et al.Heart failure and apoptosis: electrophoretic methods support data from micro- and macro-arrays. A critical review of genomics and proteomics. Proteomics.2001 Dec; 1(12):1481-8.
[38]Casey TM, Arthur PG, Bogoyevitch MA.Proteomic analysis reveals different protein changes during endothelin-1-or leukemic inhibitory factor-induced hypertrophy of cardiomyocytes in vitro. Mol Cell Proteomics.2005 May;4(5):651-61.
[39]Banfi C, Brioschi M,Wait R, et al. Proteomic analysis of membrane microdomains derived from both failing and non-failing human hearts. Proteomics,2006,6 (6):1976-1988.
[40]雷婷,赵旭宏,张曼.正常人尿蛋白质组学研究.广东医学.2010,2(31)：300-302.
[41]靳胜,张曼,张秀敏等.双向电泳技术在筛选膀胱癌特异标志蛋白中的应用.中华检验医学杂志,2006,29(5)：445-448.
[42]赵旭宏,田野,靳胜等.前列腺增生的尿液蛋白质组学分析.首都医科大学学报,2009,30(3)：277-281.
[1]Stefan Neubauer. The failing heart—an engine out of fuel. N Engl J Med,2007; 356; (11):1140-1151.
[2]Lin D, Hollander Z, Meredith A, et al.Molecular signatures of end-stage heart failure.J Card Fail.2011;17:867-74.
[3]肖志强,陈主初.疾病蛋白质组学.北京：化学工业出版社,2006 1-23.
[4]雷婷, 赵旭宏, 张曼 正常人尿蛋白质组学研究.广东医学.2010：2(31).300-302.
[5]赵亮,李祖国,丁彦青.人结肠腺癌Lovo细胞系蛋白质双向凝胶电泳技术的建立及其优化.中国现代医学杂志,2006(11)：1620-1627.
[6]Ickstein K, Cohen-Solal A, Filippatos G, et al. ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008:The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur Heart J,2008,29:2388-2442.
[7]Chung JH, Choi HJ, Kim SY,et al.Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model.BMC Genomics. 2011 Oct 20; 12:520.
[8]Haas B, Serchi T, Wagner DR,et al. Proteomic analysis of plasma samples from patients with acute myocardial infarction identifies haptoglobin as a potential prognostic biomarker.J Proteomics.2011 Dec 10;75(1):229-36.
[9]Agnetti G, Husberg C, Van Eyk JE.Divide and conquer:the application of organelle proteomics to heart failure.Circ Res.2011 Feb 18;108(4):512-26.
[10]段海峰,钱令嘉,应万涛,等.应激心肌细胞蛋白质组双向凝胶电泳分析.中国生物化学分子生物学报,2002,18(2)：234-239.
[11]Jin X, X ia L, W ang LS, et a.l D ifferen tialp rotein expressionin hypertroph ic heart w ith andw ithou t hypertens ion in spontaneous ly hypertens ive rats. Proteom ics,2006,6(6):1948-1956.
[12]Abdul S alam VB, PaulGA, AliJO, et a.l Id ent if icat ion of p lasm a p rotein b iom arkers associatedw ith id iopath ic pu lm onary arterial hypertension. Proteom ics,2006,6 (7):2286-2294.
[13]Kuznetsova T, Mischak H, Mullen W, Staessen JA. Urinary proteome analysis in hypertensive patients with left ventricular diastolic dysfunction. Eur Heart J. 2012 Sep;33(18):2342-50.
[14]Lin D, Hollander Z, Meredith A, et al.Molecular signatures of end-stage heart failure.J Card Fail.2011 Oct;17(10):867-74.
[15]Cesselli D, Beltrami AP, D'Aurizio F, et al.Effects of age and heart failure on human cardiac stem cell function.Am J Pathol.2011 Jul;179(1):349-66.
[16]Chung JH, Choi HJ, Kim SY,et al.Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model.BMC Genomics. 2011 Oct 20;12:520.
[17]Haas B, Serchi T, Wagner DR,et al. Proteomic analysis of plasma samples from patients with acute myocardial infarction identifies haptoglobin as a potential prognostic biomarker.J Proteomics.2011 Dec 10;75(1):229-36.
[18]李飞,邹亚光,毛向明等.人睾丸组织蛋白质组双向凝胶电泳技术的建立及其优化.中国男科学杂志2009；25(4)：3-6.
[19]Yin P, Zhao X, Li Q, Wang J, Li J, Xu G. Metabonomics study of intestinal fistulas based on ultraperformance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC/Q-TOF MS). Proteome Res,2006,5(9):2135-2143.
[20]赵旭宏,田野,靳胜,等.前列腺增生的尿液蛋白质组学分析[J].首都医科大学学报,2009,30(3)：277-281.
[21]靳胜,张曼,张秀敏,等.双向电泳技术在筛选膀胱癌特异标志蛋白中的应用.中华检验医学杂志,2006,29(5)：445-448.
[22]Abbaraju NV, Boutaghou MN, Townley IK, et al. Analysis of Tissue Proteomes of the Gulf Killifish, Fundulus grandis, by 2D Electrophoresis and MALDI-TOF/TOF Mass Spectrometry. Integr Comp Biol.2012 Nov;52(5):626-35.
[23]Cadete VJ, Lin HB, Sawicka J, et al.Proteomic analysis of right and left cardiac ventricles under aerobic conditions and after ischemia/reperfusion. Proteomics. 2012 Aug;12(14):2366-77.
[24]Cui Y, Zhu H, Zhu Y, et al. Proteomie analysis of testis biopies in men treated with injectable testosterone undecanoate alone or in combination with oral levonorgestrel as potential male contraceptive J Proteome Res 2008:7(9):3984-3993.
[1]Viswanathan S, Unlu M, Minden JS.Two-dimensional difference gel electrophoresis. Nat Protoc.2006;1(3):1351-8.
[2]Unlu M, Morgan ME, Minden JS. Difference gel electrophoresis:a single gel method for detecting changes in protein extracts. Electrophoresis.1997 Oct;18(11):2071-7.
[3]Wu WW, Wang G, Baek SJ, Shen RF.Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF. J Proteome Res.2006 Mar;5(3):651-8.
[4]Donahue MP, Rose K, Hochstrasser D,et al.Discovery of proteins related to coronary artery disease using industrial-scale proteomics analysis of pooled plasma. Am Heart J.2006 Sep;152(3):478-85.
[5]段海峰,钱令嘉,应万涛,等.应激心肌细胞蛋白质组双向凝胶电泳分析.中国生物化学分子生物学报,2002,18(2)：234-239.
[6]张伟英,赵猛,兰小鹏.蛋白质组学技术在心血管疾病研究中的应用.心血管康复医学杂志,2009,8(18)：403-405.
[7]Zhang J, Song MQ, Zhu JS,et,al.Identification of differentially-expressed proteins between early submucosal non-invasive and invasive colorectal cancer using 2D-DIGE and mass spectrometry. Int J Immunopathol Pharmacol.2011 Oct-Dec;24(4):849-59.
[8]Bigot P, Mouzat K,et al.Lebdai SAsian J Androl. Quantitative proteomic determination of diethylstilbestrol action on prostate cancer.2013 May;15(3):413-20.
[9]Nakagawa M, Bando Y, Nagao T, et al.Expression of p53, Ki-67, E-cadherin, N-cadherin and TOP2A in triple-negative breast cancer. Anticancer Res.2011; 1:2389-93.
[10]Grabowska MM, Day ML.Soluble E-cadherin:more than a symptom of disease. Front Biosci.2012;17:1948-64.
[11]Inge LJ, Barwe SP, D'Ambrosio J, et al.Soluble E-cadherin promotes cell survival by activating epidermal growth factor receptor. Exp Cell Res.2011;317, 838-848.
[12]Hongjuan Jiang, Guangju Guan, Rui Zhang, et al.Identification of urinary soluble E-cadherin as a novel biomarker for diabetic nephropathy. Diabetes Metab Res Rev.2009 25,232-241.
[13]Pittard AJ,Banks RE, Galley HF,et al.Soluble E-cadherin concentrations in patients with systemic inflammatory response syndrome and multiorgan dysfunction syndrome. Br J Anaesth 1996;76:629-631.
[14]Zuo JH, Zhu W, Li MY, et al.Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin. J Cell Biochem.2011; 12:2508-17.
[15]Inge LJ, Barwe SP, D'Ambrosio J,et al. Soluble E-cadherin promotes cell survival by activating epidermal growth factor receptor. Exp Cell Res.2011:317,838-848.
[16]李薇薇,袁海平,刘琼.能量限制与有氧运动对小鼠脂肪组织ZAG mRNA表达的影响.上海体育学院学报,2011,35(5)：37-41.
[17]Gao D, Trayhurn P, Bing C. Macrophage-secreted factors inhibit ZAG expression and secretion by human adipocytes. Mol Cell Endocrinol,2010,325 (1/2):135-142.
[18]Hassan MI, Waheed A, Yadav S, et al. Zinc-a2-glycoprotein:a multidisciplinary protein. Mol Cancer Res,2008,6 (6):892-906.
[19]Mracek T, Ding Q, Tzanavari T, et al. The adipokine zinc-a2-glyco-protein is downregulated with fat mass expansion in obesity. Clin Endocrinol,2010, 72(3):334-341.
[20]Jain S, Rajput A, Kumar Y, et al. Proteomic analysis of urinary protein markers for accurate prediction of diabetic kidney disorder.J Assoc Physicians India. 2005 Jun;53:513-20.
[21]Fournier T, Medjoubi N, Porquet D. Alpha-1-acid glycoprotein.Biochim. Biophys. Acta 2000; 1482:157-71.
[22]杨勇,董长林,陈广平. 血清α1-酸性糖蛋白(AAG)检测的临床价值.中国实验诊断学.2004,8(04)：382-383.
[23]Mejdoubi N, Henriques C, Bui E, Durand G, Lardeux B, Porquet D.Growth hormoneinhibits rat liver alpha-1-acid glycoprotein gene expression in vivo and in vitro.Hepatology.1999; 29(1):186-94.
[24]Christiansen MS, Hommel E, Magid E, Feldt-Rasmussen B. Orosomucoid in urine is a powerful predictor of cardiovascular mortality in normoalbuminuric patients with type 2 diabetes at five years of follow-up. Diabetologia 2005; 48: 386-93.
[25]Kobayashi H.Endogenous anti-inflammatory substances, inter-alpha-inhibitor and bikunin. Biol Chem.2006 Dec;387(12):1545-9.
[26]田国力,许洪平,董庆元,等.608例小儿尿系列微量蛋白测定及临床意义.中华肾脏病杂志,2002,18(5)：381-383.
[27]Moriguchi J, Ezaki T, Tsukahara T, et al. Effects of aging on cadmium and tubular dysfunction markers in urine from adult women in non-polluted areas [J]. Int Arch Occup Environ Health,2005,78(6):446-451.
[28]Martin H. Laboratory measurement of urine albumin and urine total protein in screening for proteinuria in chronic kidney disease.Clin Biochem Rev,2011, 32(2):97-102.
[29]谢祥鳌.几种检测肾小管功能的方法及评价.中国实用儿科杂志.2003,18(8)：451-454.
[30]Kusano E, Suzuki M,Asano Y, et al. Human alpha 1-micro-globulin and its relations hipto renal function. Neph ron; 1985,41:320-324.
[31]刘检好,冯蕴苓,高文英.儿童尿液α 1-微球蛋白检测的临床意义.河北医药.2001,23：102-103.
[32]邱晓彦,杨贵贞.恶性肿瘤细胞内存在免疫球蛋白样物质.白求恩医科大学学报,1996,22(5)：572-575.
[33]杨少波,王孟薇,龙纬缔.胃癌细胞中免疫球蛋白轻链Ig K和Igλ的共同表达.中华肿瘤杂志,2002,24(5)：465-466.
[34]Dingli D, Kyle RA, Rajkumar SV, et al. Immunoglobulin free lightchains and solitary plasm acytoma of bone. B lood,2006,108(6):1979-1983.
[35]Kyrtsonis MC,Vassilakopoulos TP,Kafasi N, et al. Prognostic value of serum free light chain ratio at diagnosis in multip lemyeloma. BrJ Haematol, 2007,137(3):240-243.
[36]vanRhee F,Bolejack V, Hollmig K, et al. High serum-free light chain levels and their rapid reduction in response to therapy definean aggressive multip lemyeloma subtype w ith poor prognosis. Blood,2007,110(3):827-832.
[37]Snozek CL, Katzmann JA, Kyle RA, et al. Prognostic value of theserum-free light chain ratio in patients with newly diagnosed myeloma and proposed incorporation into the International Staging System. Leukemia,2008,22 (10): 1933-1937.
[38]侯琳,K ousaku 0. GM2神经节苷脂沉积症发病的分子机理研究.中华医学遗传学杂志,2003,20(2)：103-105.
[39]Frey LC, Ringel SP, Folley CM. The natu ral h istory of cogn itive dysfunction in late-onset GM2 gangliosidosis. A rch Neurol,2005,62(6):989-994.
[40]Takado Y, Koid eT, Yoshikawa K, et al. A patient with GM2 gangliosidosis presenting with motorneuron disease in his forties. Rinsho Shinkeig aku, 2007,47(1):37-41.
[1]Zhang J, Song MQ, Zhu JS,et,al.Identification of differentially-expressed proteins between early submucosal non-invasive and invasive colorectal cancer using 2D-DIGE and mass spectrometry. Int J Immunopathol Pharmacol.2011 Oct-Dec;24(4):849-59.
[2]Wu WW, Wang G, Baek SJ, Shen RF.Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF. J Proteome Res.2006 Mar;5(3):651-8.
[3]Patton WF.Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci.2002 May 5;771(1-2):3-31.
[4]Hamdan M, Righetti PG.Modern strategies for protein quantification in proteome analysis:advantages and limitations. Mass Spectrom Rev.2002 Jul-Aug;21(4):287-302.
[5]Carr R, Di Bari L, Lo Piano S, Parker D, Peacock RD, Sanderson JM.A chiral probe for the acute phase proteins alpha-1-acid glycoprotein and alpha-1-antitrypsin based on europium luminescence. Dalton Trans.2012 Nov 14;41(42):13154-8.
[6]Fournier T, Medjoubi N, Porquet D. Alpha-1-acid glycoprotein.Biochim. Biophys. Acta 2000; 1482:157-71.
[7]杨勇,董长林,陈广平.血清α1-酸性糖蛋白(AAG)检测的临床价值.中国实验诊断学.2004：8(04)：382-383.
[8]Puerta A, Martin-Alvarez PJ, Ongay S, Diez-Masa JC, de Frutos M. Immunoaffinity, capillary electrophoresis, and statistics for studying intact alpha 1-acid glycoprotein isoforms as an atherothrombosis biomarker. Methods Mol Biol.2013;919:215-30.
[9]Mejdoubi N, Henriques C, Bui E, Durand G, Lardeux B, Porquet D.Growth hormoneinhibits rat liver alpha-1-acid glycoprotein gene expression in vivo and in vitro.Hepatology.1999; 29(1):186-94.
[10]Muchitsch EM, Auer W, Pichler L. Effects of alpha 1-acid glycoprotein in different rodent models of shock. Fundam Clin Pharmacol.1998; 12(2):173-81.
[11]Garcia-Munoz A, Rodriguez MA, Bologna-Molina R, et al.The orosomucoid 1 protein (al acid glycoprotein) is overexpressed in odontogenic myxoma. Proteome Sci.2012 Aug 13;10(1):49.
[12]Duche JC, Urien S, Simon N, Malaurie E, Monnet I, Barre J.Expression of the genetic variants of human alpha-1-acid glycoprotein in cancer. Clin Biochem. 2000 Apr;33(3):197-202.
[13]Lasky LA. Selectins:interpreters of cell-specific carbohydrate information during inflammation. Science.1992 Nov 6;258(5084):964-9.
[14]Ligresti G, Aplin AC, Dunn BE, Morishita A, Nicosia RF. The acute phase reactant orosomucoid-1 is a bimodal regulator of angiogenesis with time-and context-dependent inhibitory and stimulatory properties. PLoS One. 2012;7(8):e41387.
[15]Irmak S, Tilki D, Heukeshoven J, Oliveira-Ferrer L, Friedrich M, Huland H, Ergun S. Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer. Proteomics.2005 Nov;5(16):4296-304.
[16]Pang JX, Ginanni N, Dongre AR, Hefta SA, Opitek GJ. Biomarker discovery in urine by proteomics. J Proteome Res.2002 Mar-Apr;1(2):161-9.
[17]Narita T, Sasaki H, Hosoba M, Miura T,et al. Parallel increase in urinary excretion rates of immunoglobulin G, ceruloplasmin, transferrin, and orosomucoid in normoalbuminuric type 2 diabetic patients. Diabetes Care.2004 May;27(5):1176-81.
[18]Sorensson J, Matejka GL, Ohlson M, Haraldsson B. Human endothelial cells produce orosomucoid, an important component of the capillary barrier. Am. J. Physiol.1999; 276:H530-4.
[19]Jiang H, Guan G, Zhang R, Liu G, Cheng J, Hou X, Cui Y. Identification of urinary soluble E-cadherin as a novel biomarker for diabetic nephropathy. Diabetes Metab Res Rev.2009 Mar;25(3):232-41.
[20]Jiang H, Guan G, Zhang R,Increased urinary excretion of orosomucoid is a risk predictor of diabetic nephropathy. Nephrology (Carlton).2009 Apr;14(3):332-7.
[21]Christiansen MS, Hommel E, Magid E, Feldt-Rasmussen B. Orosomucoid in urine is a powerful predictor of cardiovascular mortality in normoalbuminuric patients with type 2 diabetes at five years of follow-up. Diabetologia 2005; 48: 386-93.
[2]Gheorghiade M, Bonow RO. Chronic heart failure in the United States:am an ifestation of coronary artery disease.Circulation,1998,97(3):282-289.
[3]中华医学会心血管病学分会.中国部分地区1980、1990、2000年慢性心力衰竭住院病例回顾性调查.中华心血管病杂志,2002,30(8)：450-454.
[4]Lin D, Hollander Z, Meredith A, et al.Molecular signatures of end-stage heart failure.J Card Fail.2011;17:867-74.
[5]Hunt SA, Abraham WT, Chin MH, et al. ACC/AHA 2005 guideline update for the diagnosis and management of chronic heart failure in the adult. Circulation,2005,112:1825-1852.
[6]ACC/AHA. Guidelines for the evaluation and management of chronic heart failure. Circulation,2001,104:2996-3007.
[7]顾东风,黄广勇,何江.中国心力衰竭流行病学调查及其患病率.中华心血管病杂志,2003,31(1)：03-06.
[8]AHA 2002 heart and stroke statistical update[M]. Dallas:American Heart Association,2001.1.
[9]Panagopoulou V, Deftereos S, Kossyvakis C, et al.NTproBNP:An Important Biomarker in Cardiac Diseases. Curr Top Med Chem.2013 Mar 4.
[10]Cohen-Solal.Guidelines for the diagnosis and treatment of chronic heart failure: task force for the diagnosis and treatment of chronic heart failure. Eur Heart J, 2001,22:1527-1560.
[11]McMurray JJ, Adamopoulos S, Anker SD, et al.ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012:The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC.Eur J Heart Fail.2012 Aug; 14(8):803-69.
[12]Cadete VJ, Lin HB, Sawicka J, et al.Proteomic analysis of right and left cardiac ventricles under aerobic conditions and after ischemia/reperfusion.Proteomics. 2012 Jun 11.
[13]Haas B, Serchi T, Wagner DR,et al. Proteomic analysis of plasma samples from patients with acute myocardial infarction identifies haptoglobin as a potential prognostic biomarker.Proteomics.2011 Dec 10;75(1):229-36.
[14]Wilkins MR,Williams KL,Hohstrasser DF,et al. Proteome Research:New frontiers in fuctional genomics. [M].New York:Springer,1997.
[15]Maggio ET, Ramnarayan K.Recent developments in computational proteomics. Drug Discov Today.2001 Oct 1;6(19):996-1004.
[16]O'Farrell PH.High resolution two-dimensional electrophoresis of proteins. J Biol Chem.1975 May 25;250(10):4007-21.
[17]Zhang J, Song MQ, Zhu JS,et,al.Identification of differentially-expressed proteins between early submucosal non-invasive and invasive colorectal cancer using 2D-DIGE and mass spectrometry. Int J Immunopathol Pharmacol.2011 Oct-Dec;24(4):849-59.
[18]Bigot P, Mouzat K, Lebdai S,et al. Quantitative proteomic determination of diethylstilbestrol action on prostate cancer. Asian J Androl.2013 May; 15(3):413-20.
[19]Wu WW, Wang G, Baek SJ, Shen RF.Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF. J Proteome Res.2006 Mar;5(3):651-8.
[20]成海平,钱小红.蛋白质组研究的技术体系及其进展.[J].生物化学与生物物理进展,2000,27(6)：664-667.
[21]Wright ME, Han DK, Aebersold R. Mass spectrometry-based expression profiling of clinical prostate cancer. Mol Cell Proteomics.2005 Apr;4(4):545-54.
[22]Camacho-Carvajal MM, Wollscheid B, Aebersold R,Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates:a proteomics approach. Mol Cell Proteomics.2004 Feb;3(2):176-82.
[23]Patton WF.Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci.2002 May 5;771(1-2):3-31.
[24]Hamdan M, Righetti PG. Modern strategies for protein quantification in proteome analysis:advantages and limitations. Mass Spectrom Rev.2002 Jul-Aug;21(4):287-302.
[25]Donahue MP, Rose K, Hochstrasser D,et al.Discovery of proteins related to coronary artery disease using industrial-scale proteomics analysis of pooled plasma. Am Heart J.2006 Sep;152(3):478-85.
[26]李新华,张万岱,肖冰等.蛋白质组学及其在人类疾病中的应用.中华内科杂志,2003,42(2)：133-134.
[27]张伟英,赵猛,兰小鹏.蛋白质组学技术在心血管疾病研究中的应用.心血管康复医学杂志,2009,8(18)：403-405.
[28]Weekes J, Wheeler CH, Yan JX, et, al.Bovine dilated cardiomyopathy: proteomic analysis of an animal model of human dilated cardiomyopathy. Electrophoresis.1999 Apr-May;20(4-5):898-906.
[29]De Celle T, Vanrobaeys F, Lijnen P, et al.Alterations in mouse cardiac proteome after in vivo myocardial infarction:permanent ischaemia versus ischaemia-reperfusion. Exp Physiol.2005 Jul;90(4):593-606.
[30]Jungblut PR, Zimny-Arndt U, Zeindl-Eberhart E et al.Proteomics in human disease:cancer, heart and infectious diseases.Electrophoresis.1999 Jul;20(10):2100-10.
[31]段海峰,钱令嘉,应万涛,等.应激心肌细胞蛋白质组双向凝胶电泳分析.中国生物化学分子生物学报,2002,18(2)：234-239.
[32]Jin X, X ia L, W ang LS, et a.l Differential protein expression in hypertrophic heart with and without hypertension in spontaneously hypertensive rats. Proteom ics,2006,6 (6):1948-1956.
[33]Abdul S alam VB, Pau 1 GA, A 1 i JO, et al.Identification of plasm a protein biomarkers associated with idiopathic pulmonary arterial hypertension. Proteomics,2006,6 (7):2286-2294.
[34]Zimmerli LU, Schiffer E, Zurbig P, et al.Urinary proteomic biomarkers in coronary artery disease. Mol Cell Proteomics.2008 Feb;7(2):290-8.
[35]Kuznetsova T, Mischak H, Mullen W, Staessen JA. Urinary proteome analysis in hypertensive patients with left ventricular diastolic dysfunction. Eur Heart J. 2012 Sep;33(18):2342-50.
[36]Heinke MY, Wheeler CH, Yan JX, et al.Changes in myocardial protein expression in pacing-induced canine heart failure. Electrophoresis.1999 Jul;20(10):2086-93.
[37]Jiang L, Tsubakihara M, Heinke MY,et al.Heart failure and apoptosis: electrophoretic methods support data from micro- and macro-arrays. A critical review of genomics and proteomics. Proteomics.2001 Dec; 1(12):1481-8.
[38]Casey TM, Arthur PG, Bogoyevitch MA.Proteomic analysis reveals different protein changes during endothelin-1-or leukemic inhibitory factor-induced hypertrophy of cardiomyocytes in vitro. Mol Cell Proteomics.2005 May;4(5):651-61.
[39]Banfi C, Brioschi M,Wait R, et al. Proteomic analysis of membrane microdomains derived from both failing and non-failing human hearts. Proteomics,2006,6 (6):1976-1988.
[40]雷婷,赵旭宏,张曼.正常人尿蛋白质组学研究.广东医学.2010,2(31)：300-302.
[41]靳胜,张曼,张秀敏等.双向电泳技术在筛选膀胱癌特异标志蛋白中的应用.中华检验医学杂志,2006,29(5)：445-448.
[42]赵旭宏,田野,靳胜等.前列腺增生的尿液蛋白质组学分析.首都医科大学学报,2009,30(3)：277-281.
[1]Stefan Neubauer. The failing heart—an engine out of fuel. N Engl J Med,2007; 356; (11):1140-1151.
[2]Lin D, Hollander Z, Meredith A, et al.Molecular signatures of end-stage heart failure.J Card Fail.2011;17:867-74.
[3]肖志强,陈主初.疾病蛋白质组学.北京：化学工业出版社,2006 1-23.
[4]雷婷, 赵旭宏, 张曼 正常人尿蛋白质组学研究.广东医学.2010：2(31).300-302.
[5]赵亮,李祖国,丁彦青.人结肠腺癌Lovo细胞系蛋白质双向凝胶电泳技术的建立及其优化.中国现代医学杂志,2006(11)：1620-1627.
[6]Ickstein K, Cohen-Solal A, Filippatos G, et al. ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2008:The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2008 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association of the ESC (HFA) and endorsed by the European Society of Intensive Care Medicine (ESICM). Eur Heart J,2008,29:2388-2442.
[7]Chung JH, Choi HJ, Kim SY,et al.Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model.BMC Genomics. 2011 Oct 20; 12:520.
[8]Haas B, Serchi T, Wagner DR,et al. Proteomic analysis of plasma samples from patients with acute myocardial infarction identifies haptoglobin as a potential prognostic biomarker.J Proteomics.2011 Dec 10;75(1):229-36.
[9]Agnetti G, Husberg C, Van Eyk JE.Divide and conquer:the application of organelle proteomics to heart failure.Circ Res.2011 Feb 18;108(4):512-26.
[10]段海峰,钱令嘉,应万涛,等.应激心肌细胞蛋白质组双向凝胶电泳分析.中国生物化学分子生物学报,2002,18(2)：234-239.
[11]Jin X, X ia L, W ang LS, et a.l D ifferen tialp rotein expressionin hypertroph ic heart w ith andw ithou t hypertens ion in spontaneous ly hypertens ive rats. Proteom ics,2006,6(6):1948-1956.
[12]Abdul S alam VB, PaulGA, AliJO, et a.l Id ent if icat ion of p lasm a p rotein b iom arkers associatedw ith id iopath ic pu lm onary arterial hypertension. Proteom ics,2006,6 (7):2286-2294.
[13]Kuznetsova T, Mischak H, Mullen W, Staessen JA. Urinary proteome analysis in hypertensive patients with left ventricular diastolic dysfunction. Eur Heart J. 2012 Sep;33(18):2342-50.
[14]Lin D, Hollander Z, Meredith A, et al.Molecular signatures of end-stage heart failure.J Card Fail.2011 Oct;17(10):867-74.
[15]Cesselli D, Beltrami AP, D'Aurizio F, et al.Effects of age and heart failure on human cardiac stem cell function.Am J Pathol.2011 Jul;179(1):349-66.
[16]Chung JH, Choi HJ, Kim SY,et al.Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model.BMC Genomics. 2011 Oct 20;12:520.
[17]Haas B, Serchi T, Wagner DR,et al. Proteomic analysis of plasma samples from patients with acute myocardial infarction identifies haptoglobin as a potential prognostic biomarker.J Proteomics.2011 Dec 10;75(1):229-36.
[18]李飞,邹亚光,毛向明等.人睾丸组织蛋白质组双向凝胶电泳技术的建立及其优化.中国男科学杂志2009；25(4)：3-6.
[19]Yin P, Zhao X, Li Q, Wang J, Li J, Xu G. Metabonomics study of intestinal fistulas based on ultraperformance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC/Q-TOF MS). Proteome Res,2006,5(9):2135-2143.
[20]赵旭宏,田野,靳胜,等.前列腺增生的尿液蛋白质组学分析[J].首都医科大学学报,2009,30(3)：277-281.
[21]靳胜,张曼,张秀敏,等.双向电泳技术在筛选膀胱癌特异标志蛋白中的应用.中华检验医学杂志,2006,29(5)：445-448.
[22]Abbaraju NV, Boutaghou MN, Townley IK, et al. Analysis of Tissue Proteomes of the Gulf Killifish, Fundulus grandis, by 2D Electrophoresis and MALDI-TOF/TOF Mass Spectrometry. Integr Comp Biol.2012 Nov;52(5):626-35.
[23]Cadete VJ, Lin HB, Sawicka J, et al.Proteomic analysis of right and left cardiac ventricles under aerobic conditions and after ischemia/reperfusion. Proteomics. 2012 Aug;12(14):2366-77.
[24]Cui Y, Zhu H, Zhu Y, et al. Proteomie analysis of testis biopies in men treated with injectable testosterone undecanoate alone or in combination with oral levonorgestrel as potential male contraceptive J Proteome Res 2008:7(9):3984-3993.
[1]Viswanathan S, Unlu M, Minden JS.Two-dimensional difference gel electrophoresis. Nat Protoc.2006;1(3):1351-8.
[2]Unlu M, Morgan ME, Minden JS. Difference gel electrophoresis:a single gel method for detecting changes in protein extracts. Electrophoresis.1997 Oct;18(11):2071-7.
[3]Wu WW, Wang G, Baek SJ, Shen RF.Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF. J Proteome Res.2006 Mar;5(3):651-8.
[4]Donahue MP, Rose K, Hochstrasser D,et al.Discovery of proteins related to coronary artery disease using industrial-scale proteomics analysis of pooled plasma. Am Heart J.2006 Sep;152(3):478-85.
[5]段海峰,钱令嘉,应万涛,等.应激心肌细胞蛋白质组双向凝胶电泳分析.中国生物化学分子生物学报,2002,18(2)：234-239.
[6]张伟英,赵猛,兰小鹏.蛋白质组学技术在心血管疾病研究中的应用.心血管康复医学杂志,2009,8(18)：403-405.
[7]Zhang J, Song MQ, Zhu JS,et,al.Identification of differentially-expressed proteins between early submucosal non-invasive and invasive colorectal cancer using 2D-DIGE and mass spectrometry. Int J Immunopathol Pharmacol.2011 Oct-Dec;24(4):849-59.
[8]Bigot P, Mouzat K,et al.Lebdai SAsian J Androl. Quantitative proteomic determination of diethylstilbestrol action on prostate cancer.2013 May;15(3):413-20.
[9]Nakagawa M, Bando Y, Nagao T, et al.Expression of p53, Ki-67, E-cadherin, N-cadherin and TOP2A in triple-negative breast cancer. Anticancer Res.2011; 1:2389-93.
[10]Grabowska MM, Day ML.Soluble E-cadherin:more than a symptom of disease. Front Biosci.2012;17:1948-64.
[11]Inge LJ, Barwe SP, D'Ambrosio J, et al.Soluble E-cadherin promotes cell survival by activating epidermal growth factor receptor. Exp Cell Res.2011;317, 838-848.
[12]Hongjuan Jiang, Guangju Guan, Rui Zhang, et al.Identification of urinary soluble E-cadherin as a novel biomarker for diabetic nephropathy. Diabetes Metab Res Rev.2009 25,232-241.
[13]Pittard AJ,Banks RE, Galley HF,et al.Soluble E-cadherin concentrations in patients with systemic inflammatory response syndrome and multiorgan dysfunction syndrome. Br J Anaesth 1996;76:629-631.
[14]Zuo JH, Zhu W, Li MY, et al.Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin. J Cell Biochem.2011; 12:2508-17.
[15]Inge LJ, Barwe SP, D'Ambrosio J,et al. Soluble E-cadherin promotes cell survival by activating epidermal growth factor receptor. Exp Cell Res.2011:317,838-848.
[16]李薇薇,袁海平,刘琼.能量限制与有氧运动对小鼠脂肪组织ZAG mRNA表达的影响.上海体育学院学报,2011,35(5)：37-41.
[17]Gao D, Trayhurn P, Bing C. Macrophage-secreted factors inhibit ZAG expression and secretion by human adipocytes. Mol Cell Endocrinol,2010,325 (1/2):135-142.
[18]Hassan MI, Waheed A, Yadav S, et al. Zinc-a2-glycoprotein:a multidisciplinary protein. Mol Cancer Res,2008,6 (6):892-906.
[19]Mracek T, Ding Q, Tzanavari T, et al. The adipokine zinc-a2-glyco-protein is downregulated with fat mass expansion in obesity. Clin Endocrinol,2010, 72(3):334-341.
[20]Jain S, Rajput A, Kumar Y, et al. Proteomic analysis of urinary protein markers for accurate prediction of diabetic kidney disorder.J Assoc Physicians India. 2005 Jun;53:513-20.
[21]Fournier T, Medjoubi N, Porquet D. Alpha-1-acid glycoprotein.Biochim. Biophys. Acta 2000; 1482:157-71.
[22]杨勇,董长林,陈广平. 血清α1-酸性糖蛋白(AAG)检测的临床价值.中国实验诊断学.2004,8(04)：382-383.
[23]Mejdoubi N, Henriques C, Bui E, Durand G, Lardeux B, Porquet D.Growth hormoneinhibits rat liver alpha-1-acid glycoprotein gene expression in vivo and in vitro.Hepatology.1999; 29(1):186-94.
[24]Christiansen MS, Hommel E, Magid E, Feldt-Rasmussen B. Orosomucoid in urine is a powerful predictor of cardiovascular mortality in normoalbuminuric patients with type 2 diabetes at five years of follow-up. Diabetologia 2005; 48: 386-93.
[25]Kobayashi H.Endogenous anti-inflammatory substances, inter-alpha-inhibitor and bikunin. Biol Chem.2006 Dec;387(12):1545-9.
[26]田国力,许洪平,董庆元,等.608例小儿尿系列微量蛋白测定及临床意义.中华肾脏病杂志,2002,18(5)：381-383.
[27]Moriguchi J, Ezaki T, Tsukahara T, et al. Effects of aging on cadmium and tubular dysfunction markers in urine from adult women in non-polluted areas [J]. Int Arch Occup Environ Health,2005,78(6):446-451.
[28]Martin H. Laboratory measurement of urine albumin and urine total protein in screening for proteinuria in chronic kidney disease.Clin Biochem Rev,2011, 32(2):97-102.
[29]谢祥鳌.几种检测肾小管功能的方法及评价.中国实用儿科杂志.2003,18(8)：451-454.
[30]Kusano E, Suzuki M,Asano Y, et al. Human alpha 1-micro-globulin and its relations hipto renal function. Neph ron; 1985,41:320-324.
[31]刘检好,冯蕴苓,高文英.儿童尿液α 1-微球蛋白检测的临床意义.河北医药.2001,23：102-103.
[32]邱晓彦,杨贵贞.恶性肿瘤细胞内存在免疫球蛋白样物质.白求恩医科大学学报,1996,22(5)：572-575.
[33]杨少波,王孟薇,龙纬缔.胃癌细胞中免疫球蛋白轻链Ig K和Igλ的共同表达.中华肿瘤杂志,2002,24(5)：465-466.
[34]Dingli D, Kyle RA, Rajkumar SV, et al. Immunoglobulin free lightchains and solitary plasm acytoma of bone. B lood,2006,108(6):1979-1983.
[35]Kyrtsonis MC,Vassilakopoulos TP,Kafasi N, et al. Prognostic value of serum free light chain ratio at diagnosis in multip lemyeloma. BrJ Haematol, 2007,137(3):240-243.
[36]vanRhee F,Bolejack V, Hollmig K, et al. High serum-free light chain levels and their rapid reduction in response to therapy definean aggressive multip lemyeloma subtype w ith poor prognosis. Blood,2007,110(3):827-832.
[37]Snozek CL, Katzmann JA, Kyle RA, et al. Prognostic value of theserum-free light chain ratio in patients with newly diagnosed myeloma and proposed incorporation into the International Staging System. Leukemia,2008,22 (10): 1933-1937.
[38]侯琳,K ousaku 0. GM2神经节苷脂沉积症发病的分子机理研究.中华医学遗传学杂志,2003,20(2)：103-105.
[39]Frey LC, Ringel SP, Folley CM. The natu ral h istory of cogn itive dysfunction in late-onset GM2 gangliosidosis. A rch Neurol,2005,62(6):989-994.
[40]Takado Y, Koid eT, Yoshikawa K, et al. A patient with GM2 gangliosidosis presenting with motorneuron disease in his forties. Rinsho Shinkeig aku, 2007,47(1):37-41.
[1]Zhang J, Song MQ, Zhu JS,et,al.Identification of differentially-expressed proteins between early submucosal non-invasive and invasive colorectal cancer using 2D-DIGE and mass spectrometry. Int J Immunopathol Pharmacol.2011 Oct-Dec;24(4):849-59.
[2]Wu WW, Wang G, Baek SJ, Shen RF.Comparative study of three proteomic quantitative methods, DIGE, cICAT, and iTRAQ, using 2D gel-or LC-MALDI TOF/TOF. J Proteome Res.2006 Mar;5(3):651-8.
[3]Patton WF.Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci.2002 May 5;771(1-2):3-31.
[4]Hamdan M, Righetti PG.Modern strategies for protein quantification in proteome analysis:advantages and limitations. Mass Spectrom Rev.2002 Jul-Aug;21(4):287-302.
[5]Carr R, Di Bari L, Lo Piano S, Parker D, Peacock RD, Sanderson JM.A chiral probe for the acute phase proteins alpha-1-acid glycoprotein and alpha-1-antitrypsin based on europium luminescence. Dalton Trans.2012 Nov 14;41(42):13154-8.
[6]Fournier T, Medjoubi N, Porquet D. Alpha-1-acid glycoprotein.Biochim. Biophys. Acta 2000; 1482:157-71.
[7]杨勇,董长林,陈广平.血清α1-酸性糖蛋白(AAG)检测的临床价值.中国实验诊断学.2004：8(04)：382-383.
[8]Puerta A, Martin-Alvarez PJ, Ongay S, Diez-Masa JC, de Frutos M. Immunoaffinity, capillary electrophoresis, and statistics for studying intact alpha 1-acid glycoprotein isoforms as an atherothrombosis biomarker. Methods Mol Biol.2013;919:215-30.
[9]Mejdoubi N, Henriques C, Bui E, Durand G, Lardeux B, Porquet D.Growth hormoneinhibits rat liver alpha-1-acid glycoprotein gene expression in vivo and in vitro.Hepatology.1999; 29(1):186-94.
[10]Muchitsch EM, Auer W, Pichler L. Effects of alpha 1-acid glycoprotein in different rodent models of shock. Fundam Clin Pharmacol.1998; 12(2):173-81.
[11]Garcia-Munoz A, Rodriguez MA, Bologna-Molina R, et al.The orosomucoid 1 protein (al acid glycoprotein) is overexpressed in odontogenic myxoma. Proteome Sci.2012 Aug 13;10(1):49.
[12]Duche JC, Urien S, Simon N, Malaurie E, Monnet I, Barre J.Expression of the genetic variants of human alpha-1-acid glycoprotein in cancer. Clin Biochem. 2000 Apr;33(3):197-202.
[13]Lasky LA. Selectins:interpreters of cell-specific carbohydrate information during inflammation. Science.1992 Nov 6;258(5084):964-9.
[14]Ligresti G, Aplin AC, Dunn BE, Morishita A, Nicosia RF. The acute phase reactant orosomucoid-1 is a bimodal regulator of angiogenesis with time-and context-dependent inhibitory and stimulatory properties. PLoS One. 2012;7(8):e41387.
[15]Irmak S, Tilki D, Heukeshoven J, Oliveira-Ferrer L, Friedrich M, Huland H, Ergun S. Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer. Proteomics.2005 Nov;5(16):4296-304.
[16]Pang JX, Ginanni N, Dongre AR, Hefta SA, Opitek GJ. Biomarker discovery in urine by proteomics. J Proteome Res.2002 Mar-Apr;1(2):161-9.
[17]Narita T, Sasaki H, Hosoba M, Miura T,et al. Parallel increase in urinary excretion rates of immunoglobulin G, ceruloplasmin, transferrin, and orosomucoid in normoalbuminuric type 2 diabetic patients. Diabetes Care.2004 May;27(5):1176-81.
[18]Sorensson J, Matejka GL, Ohlson M, Haraldsson B. Human endothelial cells produce orosomucoid, an important component of the capillary barrier. Am. J. Physiol.1999; 276:H530-4.
[19]Jiang H, Guan G, Zhang R, Liu G, Cheng J, Hou X, Cui Y. Identification of urinary soluble E-cadherin as a novel biomarker for diabetic nephropathy. Diabetes Metab Res Rev.2009 Mar;25(3):232-41.
[20]Jiang H, Guan G, Zhang R,Increased urinary excretion of orosomucoid is a risk predictor of diabetic nephropathy. Nephrology (Carlton).2009 Apr;14(3):332-7.
[21]Christiansen MS, Hommel E, Magid E, Feldt-Rasmussen B. Orosomucoid in urine is a powerful predictor of cardiovascular mortality in normoalbuminuric patients with type 2 diabetes at five years of follow-up. Diabetologia 2005; 48: 386-93.