發(fā)布時(shí)間：2018-04-30 03:27

  本文選題：膿毒癥 + 盲腸結(jié)扎穿刺��； 參考：《中南大學(xué)》2014年博士論文

【摘要】：膿毒癥是指由感染引起的全身炎癥反應(yīng)綜合征(systemic inflammatory response syndrome, SIRS),是重癥監(jiān)護(hù)病房(ICU)常見(jiàn)的死亡原因之一。膿毒癥的發(fā)病機(jī)制非常復(fù)雜且尚不明確,涉及患者復(fù)雜的原發(fā)病、復(fù)雜的病原體感染、復(fù)雜的毒力因子、復(fù)雜的宿主基因組多態(tài)性和機(jī)體反應(yīng)性、復(fù)雜的多系統(tǒng)相互作用網(wǎng)絡(luò)等。膿毒癥這種復(fù)雜性和非線性的特點(diǎn)為我們?cè)\斷膿毒癥和預(yù)測(cè)其預(yù)后帶來(lái)嚴(yán)重困難,也是導(dǎo)致其病死率居高不下的主要原因。近年來(lái),不少學(xué)者致力于膿毒癥生物標(biāo)志物(sepsis biomarker)研究,試圖發(fā)現(xiàn)某些可以提示膿毒癥存在與否及其嚴(yán)重程度、揭示其發(fā)病機(jī)制、區(qū)分各種微生物感染以及全身炎癥反應(yīng)和局部感染的生物標(biāo)志物,以促進(jìn)膿毒癥的診斷、預(yù)測(cè)和新的防治方法的研發(fā)。迄今為止,已發(fā)現(xiàn)超過(guò)200個(gè)生物標(biāo)志物與膿毒癥的診斷或預(yù)后預(yù)測(cè)有關(guān),但其敏感性和特異性不高,單個(gè)生物標(biāo)志物對(duì)膿毒癥的診斷、預(yù)后預(yù)測(cè)效果并不理想。目前,尚無(wú)理想的生物標(biāo)志物用于膿毒癥的臨床診斷或預(yù)后預(yù)測(cè)。因此,采用現(xiàn)代科學(xué)技術(shù),發(fā)現(xiàn)更多新的、可靠的生物標(biāo)志物以提高膿毒癥的診斷、預(yù)后預(yù)測(cè)及防治水平具有重要的科學(xué)意義。 本研究擬采用盲腸結(jié)扎穿刺(CLP)的方法來(lái)制備大鼠膿毒癥模型,并根據(jù)死亡時(shí)間將實(shí)驗(yàn)大鼠分為四組：假手術(shù)組、CLP24h死亡組、CLP48h死亡組和CLP存活組。各組大鼠于CLP術(shù)后12h通過(guò)心臟穿刺取血2-2.5ml,血液凝固后經(jīng)離心獲得血清。血清去高豐度蛋白后用iTRAQTM試劑進(jìn)行標(biāo)記,通過(guò)二維液相色譜串聯(lián)質(zhì)譜鑒定假手術(shù)對(duì)照組、膿毒癥存活組、24h死亡組和48h死亡組血清蛋白質(zhì)組的變化。采用ELISA技術(shù)對(duì)各差異表達(dá)蛋白進(jìn)行進(jìn)一步驗(yàn)證,從中篩選出新的生物標(biāo)志物,采用Logistic回歸分析方法建立膿毒癥診斷和預(yù)后預(yù)測(cè)的數(shù)學(xué)模型。同時(shí),從發(fā)現(xiàn)的生物標(biāo)志物中挑選正五聚蛋白(PTX3)用于臨床膿毒癥患者的診斷和預(yù)后預(yù)測(cè)研究,并將其結(jié)果與傳統(tǒng)的生物標(biāo)志物降鈣素原(PCT)、C反應(yīng)蛋白(CRP)進(jìn)行對(duì)比,評(píng)估其在膿毒癥患者診斷和預(yù)后判斷中的潛在意義。實(shí)驗(yàn)結(jié)果如下： 運(yùn)用iTRAQ定量蛋白質(zhì)組學(xué)方法,從膿毒癥大鼠血清中共鑒定出162個(gè)蛋白質(zhì)。其中在膿毒癥大鼠和假手術(shù)組大鼠血清中發(fā)現(xiàn)47個(gè)差異表達(dá)蛋白。與假手術(shù)組相比,膿毒癥大鼠血清中有31個(gè)蛋白質(zhì)表達(dá)升高,16個(gè)蛋白質(zhì)表達(dá)降低。在膿毒癥死亡組與存活組大鼠血清中篩選出28個(gè)差異表達(dá)蛋白。與膿毒癥大鼠存活組相比,死亡組血清中有14個(gè)蛋白質(zhì)表達(dá)升高,14個(gè)蛋白質(zhì)表達(dá)降低。 為進(jìn)一步驗(yàn)證iTRAQ定量蛋白質(zhì)組學(xué)的分析結(jié)果,我們采用ELISA方法從上述實(shí)驗(yàn)所發(fā)現(xiàn)的膿毒癥差異表達(dá)蛋白中選出25個(gè)蛋白進(jìn)一步做定量檢測(cè),發(fā)現(xiàn)其中21個(gè)蛋白質(zhì)的ELISA結(jié)果和iTRAQ結(jié)果保持一致。為篩選出對(duì)膿毒癥診斷和預(yù)后預(yù)測(cè)最有價(jià)值的生物標(biāo)志物,我們對(duì)膿毒癥大鼠和假手術(shù)組大鼠血清中25個(gè)差異表達(dá)蛋白的ELISA結(jié)果進(jìn)行了Logistic回歸分析,發(fā)現(xiàn)PTX3. MMRN1、FCN1、CPN2、PRSS1和PF4是與膿毒癥診斷密切相關(guān)的生物標(biāo)志物,根據(jù)這六個(gè)生物標(biāo)志物得出六個(gè)膿毒癥診斷相關(guān)的方程為：Logit P=1.473PTX3-37.054;Logit P=4.118MMNR1-39.249;Logit P=0.053FCN1-52.381;Logit P=0.074CPN2-21.569;Logit P=9.967PF4-6.520;Logit P=0.039PRSS1-4.227。該六個(gè)模型在大鼠膿毒癥診斷中的敏感性、特異性和準(zhǔn)確度均達(dá)到100%。同時(shí),我們將膿毒癥死亡組和存活組大鼠血清中25個(gè)差異表達(dá)蛋白的ELISA結(jié)果進(jìn)行了Logistic回歸分析,得到了一個(gè)與膿毒癥預(yù)后預(yù)測(cè)相關(guān)的模型：Logit P=-179.688+0.794MMRN1+1.682PPBP-0.003FGα-0.011FGp.采用該模型對(duì)膿毒癥大鼠的預(yù)后進(jìn)行預(yù)測(cè),發(fā)現(xiàn)其敏感性為100%,特異性為91.7%。上述研究揭示了一批潛在的新的膿毒癥生物標(biāo)志物,為膿毒癥的臨床診斷和預(yù)后預(yù)測(cè)提供了新的思路和實(shí)驗(yàn)線索。 3、為評(píng)估PTX3在膿毒癥患者診斷和預(yù)后預(yù)測(cè)中的作用,我們從ICU選擇一批膿毒癥病人進(jìn)行了臨床研究,并將PTX3與傳統(tǒng)的膿毒癥生物標(biāo)志物降鈣素原(PCT)和C-反應(yīng)蛋白(CRP)進(jìn)行了比較。結(jié)果顯示,與SIRS組相比,膿毒癥組血清PTX3、CRP和PCT濃度均升高(P0.05)；與膿毒癥存活組相比,死亡組的血清PTX3濃度進(jìn)一步升高(P0.05),而血清CRP和PCT濃度升高沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05)。血清PTX3、CRP、PCT濃度和APACHEII評(píng)分的ROC曲線分析顯示,與傳統(tǒng)的生物標(biāo)志物CRP.PCT和APACHE Ⅱ評(píng)分相比,PTX3是一個(gè)更有效的膿毒癥診斷和預(yù)后預(yù)測(cè)的生物標(biāo)志物。PTX3用于膿毒癥診斷時(shí)的ROC曲線下面積為0.963,當(dāng)其濃度高于2.43ng/ml時(shí)診斷膿毒癥的敏感性和特異性均超過(guò)90%；PTX3用于膿毒癥預(yù)后預(yù)測(cè)時(shí)的ROC曲線下面積為0.928,當(dāng)其濃度高于3.16ng/ml時(shí)預(yù)測(cè)膿毒癥預(yù)后的敏感性和特異性均超過(guò)80%。 綜上所述,本研究采用iTRAQ標(biāo)記蛋白質(zhì)組學(xué)方法篩選出一批與大鼠膿毒癥診斷和預(yù)后預(yù)測(cè)相關(guān)的生物標(biāo)志物,并選擇最佳生物標(biāo)志物建立了膿毒癥診斷和預(yù)后預(yù)測(cè)模型,發(fā)現(xiàn)了六個(gè)與膿毒癥診斷相關(guān)的生物標(biāo)志物：PTX3, MMRN1, FCN1, CPN2, PRSS1和PF4,并發(fā)現(xiàn)四個(gè)與膿毒癥預(yù)后預(yù)測(cè)相關(guān)的生物標(biāo)志物：MMRN1, PPBP, FGa和FGp。經(jīng)臨床研究發(fā)現(xiàn),PTX3作為膿毒癥診斷和預(yù)后預(yù)測(cè)的生物標(biāo)志物,其效果優(yōu)于傳統(tǒng)的生物標(biāo)志物PCT和CRP。本研究采用蛋白質(zhì)組學(xué)技術(shù),揭示了一批潛在的、新的膿毒癥生物標(biāo)志物,為膿毒癥的診斷、療效判斷及預(yù)后預(yù)測(cè)提供了新的實(shí)驗(yàn)證據(jù)和研究思路。
[Abstract]:Sepsis is the systemic inflammatory response syndrome (SIRS), which is caused by infection. It is one of the common causes of death in intensive care unit (ICU). The pathogenesis of sepsis is very complex and not clear. It involves complicated primary diseases, complex pathogens infection, complex virulence factors and complex factors. The host genome polymorphism, the organism reactivity, the complex multisystem interaction network, and so on. The complex and nonlinear characteristics of sepsis are the major causes for the diagnosis of sepsis and the prediction of its prognosis. It is also the main cause of its high mortality. In recent years, many scholars have devoted themselves to the biomarkers of sepsis. (sepsis biomarker) research, trying to find out some possible indications of the presence and severity of sepsis, revealing its pathogenesis, differentiating various microbial infections and biological markers of systemic inflammatory and local infection to promote the diagnosis, prediction and new methods of prevention and development of sepsis. So far, more than 200 have been found. The biomarkers are related to the diagnosis or prognosis prediction of sepsis, but their sensitivity and specificity are not high. Single biomarkers are not ideal for the diagnosis of sepsis and prognosis is not ideal. At present, there is no ideal biomarker for the clinical diagnosis or prognosis prediction of sepsis. Therefore, modern science and technology are used to find more new ones. Reliable biomarkers have important scientific significance in improving the diagnosis, prognosis and prevention of sepsis.
In this study, the method of cecum ligation puncture (CLP) was used to prepare rat sepsis model, and the experimental rats were divided into four groups according to the time of death: sham operation group, CLP24h death group, CLP48h death group and CLP survival group. After CLP operation, 12h was carried out by cardiac puncture and blood serum was obtained by centrifugation after blood coagulation. High abundance protein was marked with iTRAQTM reagent, and the changes of serum protein groups in sham operation control group, sepsis survival group, 24h death group and 48h death group were identified by two-dimensional liquid chromatography tandem mass spectrometry. ELISA technique was used to further verify the different expression proteins, and the new biomarkers were screened from the Logistic, and the Logistic return was used. A mathematical model for the diagnosis and prognosis of sepsis was established by the method of regression analysis. At the same time, positive five polyprotein (PTX3) was selected from the detected biomarkers for the diagnosis and prognosis of clinical sepsis, and the results were compared with the traditional biomarker calcitonin (PCT) and C reactive protein (CRP) to evaluate its sepsis. The potential significance of diagnosis and prognosis in patients with acute pancreatitis is as follows:
162 proteins were identified from the serum of sepsis rats by iTRAQ quantitative proteomic method. 47 differentially expressed proteins were found in the serum of sepsis rats and sham operation rats. Compared with the sham group, 31 proteins in the serum of sepsis rats were raised and 16 protein expressions decreased. The death of sepsis in sepsis was the death of sepsis. 28 differentially expressed proteins were screened in the sera of the group and the survival group. Compared with the survival group of the sepsis rats, the expression of 14 proteins in the serum of the death group increased and the expression of 14 proteins decreased.
In order to further verify the analysis results of iTRAQ quantitative proteomics, we selected 25 proteins from the differential expression protein of sepsis found in the above experiments to further quantitative detection, and found that the ELISA results of 21 proteins were consistent with the iTRAQ results. In order to screen out the diagnosis and prognosis of sepsis most. The ELISA results of 25 differentially expressed proteins in the serum of sepsis rats and sham operation rats were analyzed by Logistic regression analysis. We found that PTX3. MMRN1, FCN1, CPN2, PRSS1 and PF4 were biomarkers closely related to the diagnosis of sepsis. According to these six biomarkers, six sepsis were diagnosed. The related equations are: Logit P=1.473PTX3-37.054; Logit P=4.118MMNR1-39.249; Logit P=0.053FCN1-52.381; Logit P=0.074CPN2-21.569; Logit P=9.967PF4-6.520; Logit P=0.039PRSS1-4.227.. The sensitivity, specificity and accuracy of the six models in the diagnosis of rat sepsis are both at the same time, and we will be the death group of sepsis. The ELISA results of 25 differentially expressed proteins in the sera of the survival group were analyzed by Logistic regression, and a model related to the prognosis of sepsis was obtained. Logit P=-179.688+0.794MMRN1+1.682PPBP-0.003FG alpha -0.011FGp. was used to predict the prognosis of septic rats, and the sensitivity was 100% and the specificity was 9. 1.7%. these studies have revealed a number of potential new biomarkers for sepsis, which provide new ideas and experimental clues for the clinical diagnosis and prognosis prediction of sepsis.
3, in order to assess the role of PTX3 in the diagnosis and prognosis of sepsis, we conducted a clinical study from a group of ICU patients with sepsis and compared the PTX3 with the traditional septic biomarkers, calcitonin (PCT) and C- reactive protein (CRP). The results showed that the serum levels of PTX3, CRP, and PCT in the sepsis group were compared with those in the SIRS group. Compared with the survival group of sepsis (P0.05), the serum PTX3 concentration in the death group was further increased (P0.05), while the serum CRP and PCT concentrations were not statistically significant (P0.05). The ROC curve analysis of serum PTX3, CRP, PCT concentration and APACHEII score showed that compared with the traditional biomarker CRP.PCT and grade II score An effective biomarker.PTX3 for the diagnosis and prognosis of sepsis is 0.963 under the ROC curve in the diagnosis of sepsis. When its concentration is higher than 2.43ng/ml, the sensitivity and specificity of the diagnosis of sepsis are more than 90%; PTX3 is 0.928 under the ROC curve for the prognosis of sepsis, and when the concentration is higher than 3.16ng/ml The sensitivity and specificity of the sepsis test were more than 80%.
To sum up, a number of biomarkers related to the diagnosis and prognosis of sepsis in rats were screened by iTRAQ tagged proteomics, and the best biomarkers were selected to establish the diagnosis and prognosis prediction model of sepsis, and six biomarkers related to sepsis diagnosis were found: PTX3, MMRN1, FCN1, CPN2, PRSS1 and PF4, and four biomarkers associated with the prognosis prediction of sepsis: MMRN1, PPBP, FGa and FGp. have been found by clinical study. PTX3 is a biomarker for the diagnosis and prognosis prediction of sepsis. The effect is better than the traditional biomarker PCT and CRP. based proteomics technology, which reveals a potential new batch. The biomarkers of sepsis provide new experimental evidences and research ideas for the diagnosis, curative effect judgement and prognosis prediction of sepsis.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R459.7

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