风湿性瓣膜病患者血清差异多肽组学临床评估意义的研究
发布时间：2018-07-26 12:38
【摘要】：背景:从瓣膜病发生至出现临床症状,可以经历20-40年的期限,在我国瓣膜性心脏病尤其是风湿性瓣膜病仍然属于多发病,从临床症状产生至充血性心衰及活动耐量的明显下降据观察约可经历10-12年,二尖瓣狭窄较多见,合并心功能IV级的瓣膜病患者10年自然生存率低于15%。瓣膜置换术在目前仍将是治疗瓣膜病的主要手段。而心房颤动是临床上瓣膜病最常合并的心律失常之一,发生率高达60%左右,目前临床上瓣膜性心脏病外科治疗对患者远期生活质量影响较大,尤其是对已经合并心房纤颤的患者,房颤的外科治疗的远期成功率较低,而对于这类患者病情变化的整个过程中往往伴随着多种内源性多肽组份的变化,而且这些多肽组份的变化可以给我们对患者病情、心功能状态评估及干预治疗的方式选择上有很大意义。目的:通过对风湿性瓣膜病患者血清多肽组学研究,筛选出表达显著差异的多肽,探寻该差异多肽组学研究法对风湿性瓣膜病临床评估的意义。方法:本研究中对240例研究对象进行分组研究,根据美国纽约心脏病学会(NYHA)1928年心功能分级标准将240例受试对象进行分组,共分为以下三组:健康对照组即心功能正常组(不合并器质性心脏病);患者A组即心功能I-II级组;患者B组即心功能III-IV级组,每组各80名受试对象。健康组受试对象为门诊健康体检者,符合纳入排除标准,体检过程中即采集其临床指标和血清标本,患者组为住院病人,病人入院后即采集各受试对象的一般临床指标如:年龄、体重、心率、血压及心脏超声检查的相关指标如左心房直径、左心室舒张/收缩末期内径、左室射血分数、左室缩短率、相对室壁厚度、左室后壁厚度、间隔厚度及左室质量等指标,同时患者入院后次日抽取空腹静脉血,检测各研究对象的血清BNP水平,剩余血清进行多肽组学研究,每组80个血清标本中,将每16份血清各0.5μl混合成1个血清标本,每组5份混合血清标本,采用高效反向非标记定量液相质谱分析方法血清标本进行多肽组学的无标记定量分析,每个标本检测重复3次后取平均值。选择符合上述三组的5份血清样本,进行ELISA法验证丛生蛋白和载脂蛋白AI。蛋白/多肽的定量分析采用基于SWATH数据的Peakview软件进行数据定量分析,对每个样本采用强度549 m/z值进行归一化处理,数据以均数±标准差表示,统计分析应用sas9.0软件完成,两组间比较应用student'st检验分析,多肽组间差异的比较采用方差分析,p0.01为差异具有统计学意义。结果:各组研究对象在一般指标如:年龄、体重、收缩压、舒张压等,患者两组与健康组无明显统计学差异,但患者a、b组与健康组的心率分别是88.15±8.2次/分、88.19±8.5次/分、82.91±10.64次/分,患者组明显比健康组升高,患者组心率升高的原因有可能风湿性瓣膜病并发心房纤颤或者阵发性心动过速等,但患者组间比较并无明显统计学差异。超声学临床检查实验结果发现,患者a/b组的左房内径、左室舒张末内径、左室收缩末内径、射血分数、缩短率等指标分别为:(57.03±8.01)/(56.59±7.05)mm、(50.2±5.24)/(53.08±5.24)mm、(34.45±4.21)/(35.48±4.47)mm、(58.32±4.65)/(55.53±6.05)%、(32.92±4.76)/(30.65±3.69)%,健康组上述指标分别是:(31.275±2.61)mm、(44.67±6.36)mm、(29.47±3.13)mm、(68.46±1.84)%、(35.21±2.05)%,患者两组的左房内径、左室舒张末内径、左室收缩末内径明显高于健康组,但患者组间比较无明显差异,患者两组的射血分数、缩短率明显低于健康组,且患者两组间也有显著统计学差异。血清学bnp检测实验结果发现,患者a组和b组与健康组的血清bnp水平结果分别是(150.96±23.91)pg/ml、(224.96±20.49)pg/ml、(52.62±12.79)pg/ml对比有显著统计学差异,且患者a、b组间比较也有显著差异。血清多肽差异组学实验结果发现患者a、b组和健康对照组间均有38条蛋白质相关多肽和95条肽具有显著的统计学差异(校正后的p值0.0001),患者组间并未见明显统计学差异(这可能与样本量小、样本均来源于西南地区等因素有关系),这些多肽包括来自包括来自组蛋白h2b、绒毛蛋白样蛋白、补体家族c4-b、精子结构域蛋白等来源的蛋白相关多肽。其中,补体蛋白c、c4a、c4b-2、c4b结合蛋白α链均被发现在患者组明显下调;其他蛋白如α-1-酸性糖蛋白、α-2-hs糖蛋白、抗胰蛋白酶、α-1-抗胰凝乳蛋白酶、结合珠蛋白及结合珠蛋白相关肽均下调;另外,载脂蛋白ai、载脂蛋白ciii等显著下调,通过elisa检测法进行载脂蛋白ai和丛生蛋白血浆水平的验证中,载脂蛋白ai血浆水平在健康组显着降低,患者a、b组与健康组分别为(1197.82±69.57)、(1158.86±41.61)、(1414.82±98.11)μg/ml,健康组丛生蛋白血浆水平显着降低,患者a、b组与健康组分别为(306.22±14.82)、(332.72±15.83)、(177.64±7.88)μg/ml,这些差异表达的多肽表明风湿性瓣膜病根本的表现是一个动态、复杂的炎症和血栓形成过程。结论:临床上常用的心脏超声检查指标如左室内径、室壁相对厚度及射血分数、缩短分数、bnp的检测水平等能够较好的从心脏结构和功能上反映风湿性瓣膜病患者的心脏功能状态。我们通过血清差异多肽组学分析风湿性瓣膜病患者与健康人群发现了有较显著的多肽的差异表达,而这些差异的相关蛋白/多肽大部分来源于炎症和免疫反应、脂质代谢类相关蛋白,表明风湿性瓣膜病的发病是炎症和免疫反应为主伴随脂质代谢紊乱、凝血功能失衡的复杂生物反应过程,如果我们对这些差异的相关蛋白/多肽进行功能及相关通路的研究,将有助于我们理解风湿性瓣膜病的发生、发展的分子机制,同时通过差异多肽组学的研究,我们可以更深入的了解风湿性瓣膜病的演变进程,更有助于临床医师的诊治和预后判断。
[Abstract]:Background: from valvular disease to clinical symptoms, it can experience a period of 20-40 years. In our country, valvular heart disease, especially rheumatic valvular disease, still belongs to multiple diseases. It is observed from clinical symptoms to congestive heart failure and significant decrease in activity tolerance for 10-12 years. Mitral stenosis is more common, and cardiac function IV is combined. The 10 year natural survival rate of patients with valvular disease is lower than that of 15%. valve replacement, which is still the main method for the treatment of valvular disease. Atrial fibrillation is one of the most common arrhythmia in clinical valvular disease, with a rate of about 60%. The clinical treatment of valvular heart disease has a great influence on the quality of life in patients. For patients with combined atrial fibrillation, the long-term success rate of surgical treatment for atrial fibrillation is low, and the changes of endogenous polypeptide components are often accompanied by changes in the whole process of this type of patient's condition, and the changes in these polypeptide components can give us the way to the patient's disease, the assessment of heart function and the way of intervention. Objective: to search for the significance of the differential polypeptide in the clinical evaluation of rheumatic valvular disease through the study of serology of serum polypeptide in patients with rheumatic valvular disease, and to explore the significance of the differential polypeptide group in clinical evaluation of rheumatic valvular disease. Methods: in this study, 240 subjects were divided into groups, according to the New York heart disease association of the United States. NYHA) in 1928, 240 subjects were divided into three groups: the healthy control group, the normal cardiac function group (no organic heart disease), the group A, the cardiac function I-II group, the B group, the cardiac function III-IV group, and the 80 subjects in each group. The clinical indicators and serum specimens were collected during the physical examination. The patients were hospitalized and the patients were hospitalized. After admission, the general clinical indicators such as age, weight, heart rate, blood pressure, and echocardiography, such as left atrium diameter, left ventricular diastolic / end systolic diameter, left ventricular ejection score, were collected. The ratio of left ventricular shortening, relative wall thickness, left ventricular posterior wall thickness, interval thickness and left ventricular mass were measured. At the same time, the serum BNP level was detected by the patients after admission to the hospital. The residual serum was studied by polypeptides. In each group of 80 serum specimens, each of the 16 serum samples was mixed into 1 serum specimens. In each group of 5 mixed serum samples, the unmarked quantitative analysis of the serum samples was carried out by high performance reverse non labelled quantitative liquid phase mass spectrometry, and the average value of each specimen after 3 times was detected. 5 serum samples were selected in accordance with the above three groups, and the ELISA assay and apolipoprotein AI. protein / polypeptide were determined. Quantitative analysis uses Peakview software based on SWATH data to carry out quantitative analysis of data, and each sample is normalized with 549 m/z values, the data is represented by mean number of standard deviations, statistical analysis is completed by sas9.0 software, and student'st test analysis is used among the two groups, and the comparison of the differences among the polypeptides is analyzed by variance analysis, P0.01 The difference was statistically significant. Results: there was no significant difference between the two groups of the subjects in the general indexes such as age, weight, systolic pressure and diastolic pressure, but the heart rate of the two groups was 88.15 + 8.2 / min, 88.19 + 8.5 times, 82.91 + 10.64 times / scores, and the patient group was significantly higher than the health group. There was a possibility of rheumatic valvular disease complicated by atrial fibrillation or paroxysmal tachycardia in the patients with rheumatic valvular disease, but there was no significant difference between the patients. The results of the ultrasound clinical test found that the left atrium diameter, left ventricular end diastolic diameter, left ventricular end systolic diameter, ejection fraction, shortening rate, and so on were found in the a/b group. Don't be: (57.03 + 8.01) / (56.59 + 7.05) mm, (50.2 + 5.24) / (53.08 + 5.24) mm, (34.45 + 4.21) / (35.48 + 4.47) mm, (58.32 + 5.24)%. The inner diameter of the diastolic end and the left ventricular end systole was significantly higher than that of the healthy group, but there was no significant difference between the patients and the two groups. The shortening rate of the ejection fraction of the two groups was significantly lower than that of the healthy group, and there was a significant difference between the two groups. The results of serological BNP test found that the serum BNP levels of the patients and the group B and the health group were respectively, respectively. It was (150.96 + 23.91) pg/ml, (224.96 + 20.49) pg/ml and (52.62 + 12.79) pg/ml with significant statistical difference, and there was a significant difference between the patients' A and the B group. The results of serum polypeptide differential group test found that there were 38 protein related peptides and 95 peptide between the B group and the healthy control group with significant statistical differences (after correction) P value 0.0001), there was no significant statistical difference between the patients (this may be associated with the small sample size, the samples were derived from the southwest region and other factors). These peptides include protein related peptides from the sources of protein H2B, villous protein like protein, complement family c4-b, sperm domain egg white and so on. Among them, complement protein C, C4A, c4b- 2, C4B binding protein alpha chain was found to be significantly downregulated in the patient group; other proteins, such as alpha -1- acid glycoprotein, alpha -2-hs glycoprotein, anti trypsin, alpha -1- anti chymotrypsin, binding globin and globin related peptide, were downregulated, and apolipoprotein AI, apolipoprotein CIII, and so on were significantly downregulated by ELISA detection of apolipoprotein. The plasma levels of apolipoprotein AI and AI were significantly decreased in the healthy group, and in the patients with a, the group B and the healthy group were (1197.82 + 69.57), (1158.86 + 41.61), (1414.82 + 98.11) mu g/ml, the plasma level of the healthy group decreased significantly, the patients a, B group and the healthy group (332.72 + 15.83), (332.72 + 15.83), (177. 15.83), respectively, (332.72 + 15.83), (332.72 + 15.83), (332.72 + 15.83), respectively, (332.72 + 15.83), (332.72 + 15.83), respectively, (332.72 + 15.83), (332.72 + 15.83), respectively, (332.72 + 15.83), respectively, (332.72 + 15.83), (177., 15.83), respectively. 64 + 7.88) mu g/ml, these differentially expressed peptides indicate that the fundamental manifestation of rheumatic valvular disease is a dynamic, complicated process of inflammation and thrombosis. Conclusion: the commonly used echocardiographic indexes, such as the left ventricular diameter, the relative thickness of the ventricular wall and the ejection fraction, the number of shortening, the level of the detection of BNP, can be better from the heart structure. The functional state of the heart function in patients with rheumatic valvular disease. We found a significant difference in the expression of polypeptide in patients with rheumatic valvular disease and healthy people through serum differential polypeptide group, and most of these differences were derived from inflammatory and immune responses, and lipid metabolism related proteins. The pathogenesis of rheumatic valvular valvular disease is the complex bioreactive process of inflammation and immune response associated with lipid metabolism disorder and coagulation dysfunction. If we study the function and related pathways of these proteins / peptides, it will help us to understand the pathogenesis of rheumatic valvular disease and the molecular mechanism of development. Through the study of differential proteomics, we can have a better understanding of the evolution of rheumatic valvular disease and help clinicians in the diagnosis and treatment of prognosis.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.2
本文编号：2146039
[Abstract]:Background: from valvular disease to clinical symptoms, it can experience a period of 20-40 years. In our country, valvular heart disease, especially rheumatic valvular disease, still belongs to multiple diseases. It is observed from clinical symptoms to congestive heart failure and significant decrease in activity tolerance for 10-12 years. Mitral stenosis is more common, and cardiac function IV is combined. The 10 year natural survival rate of patients with valvular disease is lower than that of 15%. valve replacement, which is still the main method for the treatment of valvular disease. Atrial fibrillation is one of the most common arrhythmia in clinical valvular disease, with a rate of about 60%. The clinical treatment of valvular heart disease has a great influence on the quality of life in patients. For patients with combined atrial fibrillation, the long-term success rate of surgical treatment for atrial fibrillation is low, and the changes of endogenous polypeptide components are often accompanied by changes in the whole process of this type of patient's condition, and the changes in these polypeptide components can give us the way to the patient's disease, the assessment of heart function and the way of intervention. Objective: to search for the significance of the differential polypeptide in the clinical evaluation of rheumatic valvular disease through the study of serology of serum polypeptide in patients with rheumatic valvular disease, and to explore the significance of the differential polypeptide group in clinical evaluation of rheumatic valvular disease. Methods: in this study, 240 subjects were divided into groups, according to the New York heart disease association of the United States. NYHA) in 1928, 240 subjects were divided into three groups: the healthy control group, the normal cardiac function group (no organic heart disease), the group A, the cardiac function I-II group, the B group, the cardiac function III-IV group, and the 80 subjects in each group. The clinical indicators and serum specimens were collected during the physical examination. The patients were hospitalized and the patients were hospitalized. After admission, the general clinical indicators such as age, weight, heart rate, blood pressure, and echocardiography, such as left atrium diameter, left ventricular diastolic / end systolic diameter, left ventricular ejection score, were collected. The ratio of left ventricular shortening, relative wall thickness, left ventricular posterior wall thickness, interval thickness and left ventricular mass were measured. At the same time, the serum BNP level was detected by the patients after admission to the hospital. The residual serum was studied by polypeptides. In each group of 80 serum specimens, each of the 16 serum samples was mixed into 1 serum specimens. In each group of 5 mixed serum samples, the unmarked quantitative analysis of the serum samples was carried out by high performance reverse non labelled quantitative liquid phase mass spectrometry, and the average value of each specimen after 3 times was detected. 5 serum samples were selected in accordance with the above three groups, and the ELISA assay and apolipoprotein AI. protein / polypeptide were determined. Quantitative analysis uses Peakview software based on SWATH data to carry out quantitative analysis of data, and each sample is normalized with 549 m/z values, the data is represented by mean number of standard deviations, statistical analysis is completed by sas9.0 software, and student'st test analysis is used among the two groups, and the comparison of the differences among the polypeptides is analyzed by variance analysis, P0.01 The difference was statistically significant. Results: there was no significant difference between the two groups of the subjects in the general indexes such as age, weight, systolic pressure and diastolic pressure, but the heart rate of the two groups was 88.15 + 8.2 / min, 88.19 + 8.5 times, 82.91 + 10.64 times / scores, and the patient group was significantly higher than the health group. There was a possibility of rheumatic valvular disease complicated by atrial fibrillation or paroxysmal tachycardia in the patients with rheumatic valvular disease, but there was no significant difference between the patients. The results of the ultrasound clinical test found that the left atrium diameter, left ventricular end diastolic diameter, left ventricular end systolic diameter, ejection fraction, shortening rate, and so on were found in the a/b group. Don't be: (57.03 + 8.01) / (56.59 + 7.05) mm, (50.2 + 5.24) / (53.08 + 5.24) mm, (34.45 + 4.21) / (35.48 + 4.47) mm, (58.32 + 5.24)%. The inner diameter of the diastolic end and the left ventricular end systole was significantly higher than that of the healthy group, but there was no significant difference between the patients and the two groups. The shortening rate of the ejection fraction of the two groups was significantly lower than that of the healthy group, and there was a significant difference between the two groups. The results of serological BNP test found that the serum BNP levels of the patients and the group B and the health group were respectively, respectively. It was (150.96 + 23.91) pg/ml, (224.96 + 20.49) pg/ml and (52.62 + 12.79) pg/ml with significant statistical difference, and there was a significant difference between the patients' A and the B group. The results of serum polypeptide differential group test found that there were 38 protein related peptides and 95 peptide between the B group and the healthy control group with significant statistical differences (after correction) P value 0.0001), there was no significant statistical difference between the patients (this may be associated with the small sample size, the samples were derived from the southwest region and other factors). These peptides include protein related peptides from the sources of protein H2B, villous protein like protein, complement family c4-b, sperm domain egg white and so on. Among them, complement protein C, C4A, c4b- 2, C4B binding protein alpha chain was found to be significantly downregulated in the patient group; other proteins, such as alpha -1- acid glycoprotein, alpha -2-hs glycoprotein, anti trypsin, alpha -1- anti chymotrypsin, binding globin and globin related peptide, were downregulated, and apolipoprotein AI, apolipoprotein CIII, and so on were significantly downregulated by ELISA detection of apolipoprotein. The plasma levels of apolipoprotein AI and AI were significantly decreased in the healthy group, and in the patients with a, the group B and the healthy group were (1197.82 + 69.57), (1158.86 + 41.61), (1414.82 + 98.11) mu g/ml, the plasma level of the healthy group decreased significantly, the patients a, B group and the healthy group (332.72 + 15.83), (332.72 + 15.83), (177. 15.83), respectively, (332.72 + 15.83), (332.72 + 15.83), (332.72 + 15.83), respectively, (332.72 + 15.83), (332.72 + 15.83), respectively, (332.72 + 15.83), (332.72 + 15.83), respectively, (332.72 + 15.83), respectively, (332.72 + 15.83), (177., 15.83), respectively. 64 + 7.88) mu g/ml, these differentially expressed peptides indicate that the fundamental manifestation of rheumatic valvular disease is a dynamic, complicated process of inflammation and thrombosis. Conclusion: the commonly used echocardiographic indexes, such as the left ventricular diameter, the relative thickness of the ventricular wall and the ejection fraction, the number of shortening, the level of the detection of BNP, can be better from the heart structure. The functional state of the heart function in patients with rheumatic valvular disease. We found a significant difference in the expression of polypeptide in patients with rheumatic valvular disease and healthy people through serum differential polypeptide group, and most of these differences were derived from inflammatory and immune responses, and lipid metabolism related proteins. The pathogenesis of rheumatic valvular valvular disease is the complex bioreactive process of inflammation and immune response associated with lipid metabolism disorder and coagulation dysfunction. If we study the function and related pathways of these proteins / peptides, it will help us to understand the pathogenesis of rheumatic valvular disease and the molecular mechanism of development. Through the study of differential proteomics, we can have a better understanding of the evolution of rheumatic valvular disease and help clinicians in the diagnosis and treatment of prognosis.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.2
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