發(fā)布時間：2018-03-10 15:03

  本文選題：蛋白質組學　切入點：先天性心臟病　出處：《天津醫(yī)科大學》2014年博士論文　論文類型：學位論文

【摘要】：目的：一、本研究選擇單純性室間隔缺損(Ventricular Septal Defect, VSD)患兒作為研究對象,假設其血漿中某些蛋白質的表達受疾病影響而發(fā)生改變,以健康兒童為對照,通過蛋白質組學技術尋找并確定VSD患兒血漿中的特異性差異蛋白質。二、依據(jù)VSD的病理特點,分析和探討差異蛋白質潛在的臨床意義；希望找到VSD的特異性蛋白質作為篩查的生物標記物。三、探討和總結蛋白質組學方法在先天性心臟病(Congenital Heart Disease, CHD)中的研究方法和路線,為蛋白質組學技術今后在復雜CHD研究中的應用奠定基礎。 方法：一、選取55例連續(xù)的接受外科手術治療的VSD患兒作為研究對象,同期健康兒童55例組成對照組。于術前晨起空腹抽靜脈血2m1,置于EDTA抗凝采血管中,離心后收集血漿,于-80℃C超低溫冰箱內儲存。先由VSD組和對照組分別隨機選取15例患兒血漿,充分混合后應用白蛋白/IgG去除試劑盒去除血漿中的高豐度蛋白,在進行樣品血漿蛋白的稀釋定量之后,將兩組的血漿樣本行雙向電泳(2-DE),每組樣本均重復進行2-DE3次,通過考馬斯亮藍凝膠染色和圖像掃描選取差異蛋白質點。對差異蛋白質點進行基質輔助激光解吸離子化-飛行時間質譜(MALDI-TOFMS)鑒定,得到蛋白質點的肽質量指紋圖譜(PMF),然后利用GPS軟件,在Swissprot蛋白數(shù)據(jù)庫中搜索確定蛋白質種類。二、取另外40例VSD組和40例對照組患兒血漿,對鑒定出的差異蛋白質進行ELISA實驗定量驗證,以明確其表達變化與疾病是否存在顯著關聯(lián)。每個樣本重復測量3次,計算其平均吸光值,根據(jù)標準曲線確定樣品濃度,實驗組和對照組之間的濃度差異應用非配對t檢驗進行統(tǒng)計,P0.05則認為差異顯著。 結果：一、2-DE的結果經GS-800圖像采集和PD-Quest軟件分析,在VSD組中分別檢測到蛋白點325個,320個和317個,匹配率97.25%；在對照組中分別檢測到蛋白點318個,313個和316個,匹配率98.11%。篩選確定差異蛋白位點4個,其中3個表達下調,1個表達上調。經MALDI-TOF質譜鑒定,表達下調的蛋白質為血清淀粉樣蛋白P (SAP)、觸珠蛋白(Hp)、纖維膠凝蛋白-3(Ficolin-3),表達上調蛋白質為α-1-酸性糖蛋白(α1-AGP)。二、40例樣本的VSD組ELISA實驗測定血漿SAP濃度為3.818±0.2294ng/ml,Hp濃度為0.3965±0.04186mg/ml, Ficolin-3濃度為5.548±0.343ug/ml, α1-AGP濃度為3.086±0.1294mg/ml;40例健康兒童對照組血漿SAP濃度為6.270±0.7723ng/ml,Hp濃度為0.5994±0.07097mg/ml, Ficolin-3濃度為10.576±1.576ug/ml, α,-AGP濃度為2.302±0.1071mg/ml。與對照組相比,VSD組患兒血漿中SAP呈顯著低表達(P=0.0029),Hp呈顯著低表達(P=0.016),Ficolin-3呈顯著低表達(P=0.003),α1-AGP呈顯著高表達(P0.0001),以上差異均具有統(tǒng)計學意義,并與2-DE測定出的結果相符。 結論：本研究通過蛋白質組技術,在VSD患兒與健康人群之間發(fā)現(xiàn)了4種血漿差異蛋白質；并經更大樣本的ELISA實驗證實。其中SAP表達下調提示VSD患兒的免疫應答與炎癥反應發(fā)生了變化。Hp表達下調則與VSD患兒對呼吸道感染的抵抗力下降和容易并發(fā)肺動脈高壓有關。Ficolin-3的下調可能與VSD患兒易患呼吸道感染有關,而且提示VSD患者可能存在FCN3基因缺陷。而表達上調的α1-AGP可能與VSD患兒的相對應激狀態(tài)有關,其有可能作為評判VSD患兒應激強度和選擇手術時機的生物標記物。差異蛋白質的發(fā)現(xiàn)對于深入了解VSD的病理特點具有實際意義,而且為進一步的機制研究和生物標記物研究指明了方向。本研究也顯示出蛋白質組學技術在CHD研究中的價值,為今后在復雜CHD研究中的應用奠定了基礎。
[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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R726.5

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