本文選題：蛋白質(zhì)組學(xué)　切入點(diǎn)：先天性心臟病　出處：《天津醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)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(shè)其血漿中某些蛋白質(zhì)的表達(dá)受疾病影響而發(fā)生改變,以健康兒童為對照,通過蛋白質(zhì)組學(xué)技術(shù)尋找并確定VSD患兒血漿中的特異性差異蛋白質(zhì)。二、依據(jù)VSD的病理特點(diǎn),分析和探討差異蛋白質(zhì)潛在的臨床意義；希望找到VSD的特異性蛋白質(zhì)作為篩查的生物標(biāo)記物。三、探討和總結(jié)蛋白質(zhì)組學(xué)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(zhì)組學(xué)技術(shù)今后在復(fù)雜CHD研究中的應(yīng)用奠定基礎(chǔ)。 方法：一、選取55例連續(xù)的接受外科手術(shù)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(shù)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(nèi)儲存。先由VSD組和對照組分別隨機(jī)選取15例患兒血漿,充分混合后應(yīng)用白蛋白/IgG去除試劑盒去除血漿中的高豐度蛋白,在進(jìn)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(fù)進(jìn)行2-DE3次,通過考馬斯亮藍(lán)凝膠染色和圖像掃描選取差異蛋白質(zhì)點(diǎn)。對差異蛋白質(zhì)點(diǎn)進(jìn)行基質(zhì)輔助激光解吸離子化-飛行時間質(zhì)譜(MALDI-TOFMS)鑒定,得到蛋白質(zhì)點(diǎn)的肽質(zhì)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(zhì)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(zhì)進(jìn)行ELISA實驗定量驗證,以明確其表達(dá)變化與疾病是否存在顯著關(guān)聯(lián)。每個樣本重復(fù)測量3次,計算其平均吸光值,根據(jù)標(biāo)準(zhǔn)曲線確定樣品濃度,實驗組和對照組之間的濃度差異應(yīng)用非配對t檢驗進(jìn)行統(tǒng)計,P0.05則認(rèn)為差異顯著。 結(jié)果：一、2-DE的結(jié)果經(jīng)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(diǎn)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(diǎn)318個,313個和316個,匹配率98.11%。篩選確定差異蛋白位點(diǎn)4個,其中3個表達(dá)下調(diào),1個表達(dá)上調(diào)。經(jīng)MALDI-TOF質(zhì)譜鑒定,表達(dá)下調(diào)的蛋白質(zhì)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(dá)上調(diào)蛋白質(zhì)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(dá)(P=0.0029),Hp呈顯著低表達(dá)(P=0.016),Ficolin-3呈顯著低表達(dá)(P=0.003),α1-AGP呈顯著高表達(dá)(P0.0001),以上差異均具有統(tǒng)計學(xué)意義,并與2-DE測定出的結(jié)果相符。 結(jié)論：本研究通過蛋白質(zhì)組技術(shù),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(zhì)；并經(jīng)更大樣本的ELISA實驗證實。其中SAP表達(dá)下調(diào)提示VSD患兒的免疫應(yīng)答與炎癥反應(yīng)發(fā)生了變化。Hp表達(dá)下調(diào)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(guān)。Ficolin-3的下調(diào)可能與VSD患兒易患呼吸道感染有關(guān),而且提示VSD患者可能存在FCN3基因缺陷。而表達(dá)上調(diào)的α1-AGP可能與VSD患兒的相對應(yīng)激狀態(tài)有關(guān),其有可能作為評判VSD患兒應(yīng)激強(qiáng)度和選擇手術(shù)時機(jī)的生物標(biāo)記物。差異蛋白質(zhì)的發(fā)現(xiàn)對于深入了解VSD的病理特點(diǎn)具有實際意義,而且為進(jìn)一步的機(jī)制研究和生物標(biāo)記物研究指明了方向。本研究也顯示出蛋白質(zhì)組學(xué)技術(shù)在CHD研究中的價值,為今后在復(fù)雜CHD研究中的應(yīng)用奠定了基礎(chǔ)。
[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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R726.5

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