| BackgroudChronic 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 patientsObjectiveTo 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 MethodsRandom 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.Results1. 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.ConclusionUrine 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 failureObjectiveUsing2D-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 MethodsThe 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.Results1. 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).ConclusionSuccessful 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 failureObjectiveTo 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 MethodsFirst, 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.Results1. 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.ConclusionThe 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. |
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