单纯性室间隔缺损的血浆差异蛋白质组学研究

发布时间：2018-03-10 15:03

本文选题：蛋白质组学　切入点：先天性心脏病　出处：《天津医科大学》2014年博士论文　论文类型：学位论文

【摘要】：目的：一、本研究选择单纯性室间隔缺损(Ventricular Septal Defect, VSD)患儿作为研究对象,假设其血浆中某些蛋白质的表达受疾病影响而发生改变,以健康儿童为对照,通过蛋白质组学技术寻找并确定VSD患儿血浆中的特异性差异蛋白质。二、依据VSD的病理特点,分析和探讨差异蛋白质潜在的临床意义；希望找到VSD的特异性蛋白质作为筛查的生物标记物。三、探讨和总结蛋白质组学方法在先天性心脏病(Congenital Heart Disease, CHD)中的研究方法和路线,为蛋白质组学技术今后在复杂CHD研究中的应用奠定基础。方法：一、选取55例连续的接受外科手术治疗的VSD患儿作为研究对象,同期健康儿童55例组成对照组。于术前晨起空腹抽静脉血2m1,置于EDTA抗凝采血管中,离心后收集血浆,于-80℃C超低温冰箱内储存。先由VSD组和对照组分别随机选取15例患儿血浆,充分混合后应用白蛋白/IgG去除试剂盒去除血浆中的高丰度蛋白,在进行样品血浆蛋白的稀释定量之后,将两组的血浆样本行双向电泳(2-DE),每组样本均重复进行2-DE3次,通过考马斯亮蓝凝胶染色和图像扫描选取差异蛋白质点。对差异蛋白质点进行基质辅助激光解吸离子化-飞行时间质谱(MALDI-TOFMS)鉴定,得到蛋白质点的肽质量指纹图谱(PMF),然后利用GPS软件,在Swissprot蛋白数据库中搜索确定蛋白质种类。二、取另外40例VSD组和40例对照组患儿血浆,对鉴定出的差异蛋白质进行ELISA实验定量验证,以明确其表达变化与疾病是否存在显著关联。每个样本重复测量3次,计算其平均吸光值,根据标准曲线确定样品浓度,实验组和对照组之间的浓度差异应用非配对t检验进行统计,P0.05则认为差异显著。结果：一、2-DE的结果经GS-800图像采集和PD-Quest软件分析,在VSD组中分别检测到蛋白点325个,320个和317个,匹配率97.25%；在对照组中分别检测到蛋白点318个,313个和316个,匹配率98.11%。筛选确定差异蛋白位点4个,其中3个表达下调,1个表达上调。经MALDI-TOF质谱鉴定,表达下调的蛋白质为血清淀粉样蛋白P (SAP)、触珠蛋白(Hp)、纤维胶凝蛋白-3(Ficolin-3),表达上调蛋白质为α-1-酸性糖蛋白(α1-AGP)。二、40例样本的VSD组ELISA实验测定血浆SAP浓度为3.818±0.2294ng/ml,Hp浓度为0.3965±0.04186mg/ml, Ficolin-3浓度为5.548±0.343ug/ml, α1-AGP浓度为3.086±0.1294mg/ml;40例健康儿童对照组血浆SAP浓度为6.270±0.7723ng/ml,Hp浓度为0.5994±0.07097mg/ml, Ficolin-3浓度为10.576±1.576ug/ml, α,-AGP浓度为2.302±0.1071mg/ml。与对照组相比,VSD组患儿血浆中SAP呈显著低表达(P=0.0029),Hp呈显著低表达(P=0.016),Ficolin-3呈显著低表达(P=0.003),α1-AGP呈显著高表达(P0.0001),以上差异均具有统计学意义,并与2-DE测定出的结果相符。结论：本研究通过蛋白质组技术,在VSD患儿与健康人群之间发现了4种血浆差异蛋白质；并经更大样本的ELISA实验证实。其中SAP表达下调提示VSD患儿的免疫应答与炎症反应发生了变化。Hp表达下调则与VSD患儿对呼吸道感染的抵抗力下降和容易并发肺动脉高压有关。Ficolin-3的下调可能与VSD患儿易患呼吸道感染有关,而且提示VSD患者可能存在FCN3基因缺陷。而表达上调的α1-AGP可能与VSD患儿的相对应激状态有关,其有可能作为评判VSD患儿应激强度和选择手术时机的生物标记物。差异蛋白质的发现对于深入了解VSD的病理特点具有实际意义,而且为进一步的机制研究和生物标记物研究指明了方向。本研究也显示出蛋白质组学技术在CHD研究中的价值,为今后在复杂CHD研究中的应用奠定了基础。
[Abstract]:Objective: in this study, ventricular septal defect (Ventricular Septal Defect, VSD) were selected as the research object, the expression of some proteins in plasma that affected changes in healthy children as control by proteomic technology to find and identify specific differences in protein level in children with VSD in two. And according to the pathological characteristics of VSD, analyze and discuss the clinical significance of differences in protein potential; to find the specific protein VSD as a biomarker screening. Three, explore and summarize the method of proteomics in congenital heart disease (Congenital Heart, Disease, CHD) research methods and routes, to lay the foundation for application in the study of complex CHD in the future for proteomics.
Methods: a selected 55 consecutive patients underwent surgical treatment in children with VSD as the research object, the same period 55 cases of healthy children as control group. Preoperative fasting venous blood 2M1, EDTA in blood anticoagulant, after centrifugation, plasma was collected at -80 DEG C C ultra low temperature freezer stored in the VSD. Group and control group were randomly selected 15 cases of plasma, after mixing with albumin removal kit /IgG removal of high abundance proteins in plasma, after dilution quantitative samples of plasma proteins, two groups of plasma samples for two-dimensional electrophoresis (2-DE), each group of samples were repeated for 2-DE3 times, by Coomassie blue gel staining and image scanning. The result of matrix assisted laser desorption ionization time-of-flight mass spectrometry of differential proteins (MALDI-TOFMS) identification, peptide mass fingerprint of protein spots (PMF), and After using the GPS software in the Swissprot protein database search to determine the types of protein. Two, other 40 cases of VSD group and 40 cases in control group plasma ELISA experimental verification of quantitative protein identified differentially, to determine the expression and the presence of disease significantly related. Each sample is 3 times of repeated measurement, calculation the average absorbance value, determination of the concentration of the sample according to the standard curve between the experimental group and the control group the concentration differences using the unpaired t test statistics, P0.05 believes that the difference was significant.
Results: the results of 2-DE, by GS-800 PD-Quest image acquisition and analysis software, in the VSD group were detected protein spots 325, 320 and 317, the matching rate is 97.25%; in the control group were detected protein spots 318, 313 and 316, the matching rate of 98.11%. screening to determine the difference in protein 4 sites, among which 3 were down regulated and 1 up-regulated. Identified by MALDI-TOF mass spectrometry, expression of protein and serum amyloid P protein (SAP), haptoglobin (Hp), gel fiber protein -3 (Ficolin-3), expression of protein -1- alpha acid glycoprotein (alpha 1-AGP) two, 40 cases of VSD group ELISA test samples for determination of plasma SAP concentration was 3.818 + 0.2294ng/ml, the concentration of Hp was 0.3965 + 0.04186mg/ml, the concentration of Ficolin-3 was 5.548 + 0.343ug/ml, alpha 1-AGP concentration was 3.086 + 0.1294mg/ml; 40 cases of healthy children in control group the plasma concentration of SAP was 6.270 + 0.7723ng/ml, the concentration of Hp was 0.5994 The concentration of Ficolin-3 + 0.07097mg/ml, 10.576 + 1.576ug/ml, alpha, -AGP concentration was 2.302 + 0.1071mg/ml. compared with the control group, the expression of SAP was statistically lower in VSD group (P=0.0029), plasma Hp was significantly lower (P=0.016), the expression of Ficolin-3 was significantly low expression (P=0.003), alpha expression of 1-AGP was significantly higher (P0.0001) above, the difference was statistically significant, and 2-DE measured results.
Conclusion: This study by proteomic techniques, 4 plasma proteins were found in between VSD patients and healthy people; and by the ELISA experiment of larger samples confirmed. The decreased expression of SAP immune response and inflammation that VSD children have changed the downregulation of the expression of.Hp and VSD in children with respiratory tract infection resistance drops and easily complicated with pulmonary arterial hypertension related to downregulation of.Ficolin-3 and VSD may be susceptible to respiratory tract infection of children, and suggest that VSD patients may have FCN3 gene defect. The relative stress and the expression of alpha 1-AGP increased the possibility of children with VSD, which may serve as biomarkers for evaluation of children with VSD stress intensity and timing of operation. The difference of protein has practical significance for in-depth understanding of the pathological features of VSD, and for the study of mechanisms and biomarkers further specified This study also shows the value of proteomics technology in CHD research, which lays the foundation for future application in complex CHD research.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R726.5

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