基于蛋白质组学策略的脓毒症生物标志物研究

发布时间：2018-04-30 03:27

本文选题：脓毒症 + 盲肠结扎穿刺　；参考：《中南大学》2014年博士论文

【摘要】：脓毒症是指由感染引起的全身炎症反应综合征(systemic inflammatory response syndrome, SIRS),是重症监护病房(ICU)常见的死亡原因之一。脓毒症的发病机制非常复杂且尚不明确,涉及患者复杂的原发病、复杂的病原体感染、复杂的毒力因子、复杂的宿主基因组多态性和机体反应性、复杂的多系统相互作用网络等。脓毒症这种复杂性和非线性的特点为我们诊断脓毒症和预测其预后带来严重困难,也是导致其病死率居高不下的主要原因。近年来,不少学者致力于脓毒症生物标志物(sepsis biomarker)研究,试图发现某些可以提示脓毒症存在与否及其严重程度、揭示其发病机制、区分各种微生物感染以及全身炎症反应和局部感染的生物标志物,以促进脓毒症的诊断、预测和新的防治方法的研发。迄今为止,已发现超过200个生物标志物与脓毒症的诊断或预后预测有关,但其敏感性和特异性不高,单个生物标志物对脓毒症的诊断、预后预测效果并不理想。目前,尚无理想的生物标志物用于脓毒症的临床诊断或预后预测。因此,采用现代科学技术,发现更多新的、可靠的生物标志物以提高脓毒症的诊断、预后预测及防治水平具有重要的科学意义。本研究拟采用盲肠结扎穿刺(CLP)的方法来制备大鼠脓毒症模型,并根据死亡时间将实验大鼠分为四组：假手术组、CLP24h死亡组、CLP48h死亡组和CLP存活组。各组大鼠于CLP术后12h通过心脏穿刺取血2-2.5ml,血液凝固后经离心获得血清。血清去高丰度蛋白后用iTRAQTM试剂进行标记,通过二维液相色谱串联质谱鉴定假手术对照组、脓毒症存活组、24h死亡组和48h死亡组血清蛋白质组的变化。采用ELISA技术对各差异表达蛋白进行进一步验证,从中筛选出新的生物标志物,采用Logistic回归分析方法建立脓毒症诊断和预后预测的数学模型。同时,从发现的生物标志物中挑选正五聚蛋白(PTX3)用于临床脓毒症患者的诊断和预后预测研究,并将其结果与传统的生物标志物降钙素原(PCT)、C反应蛋白(CRP)进行对比,评估其在脓毒症患者诊断和预后判断中的潜在意义。实验结果如下：运用iTRAQ定量蛋白质组学方法,从脓毒症大鼠血清中共鉴定出162个蛋白质。其中在脓毒症大鼠和假手术组大鼠血清中发现47个差异表达蛋白。与假手术组相比,脓毒症大鼠血清中有31个蛋白质表达升高,16个蛋白质表达降低。在脓毒症死亡组与存活组大鼠血清中筛选出28个差异表达蛋白。与脓毒症大鼠存活组相比,死亡组血清中有14个蛋白质表达升高,14个蛋白质表达降低。为进一步验证iTRAQ定量蛋白质组学的分析结果,我们采用ELISA方法从上述实验所发现的脓毒症差异表达蛋白中选出25个蛋白进一步做定量检测,发现其中21个蛋白质的ELISA结果和iTRAQ结果保持一致。为筛选出对脓毒症诊断和预后预测最有价值的生物标志物,我们对脓毒症大鼠和假手术组大鼠血清中25个差异表达蛋白的ELISA结果进行了Logistic回归分析,发现PTX3. MMRN1、FCN1、CPN2、PRSS1和PF4是与脓毒症诊断密切相关的生物标志物,根据这六个生物标志物得出六个脓毒症诊断相关的方程为：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。该六个模型在大鼠脓毒症诊断中的敏感性、特异性和准确度均达到100%。同时,我们将脓毒症死亡组和存活组大鼠血清中25个差异表达蛋白的ELISA结果进行了Logistic回归分析,得到了一个与脓毒症预后预测相关的模型：Logit P=-179.688+0.794MMRN1+1.682PPBP-0.003FGα-0.011FGp.采用该模型对脓毒症大鼠的预后进行预测,发现其敏感性为100%,特异性为91.7%。上述研究揭示了一批潜在的新的脓毒症生物标志物,为脓毒症的临床诊断和预后预测提供了新的思路和实验线索。 3、为评估PTX3在脓毒症患者诊断和预后预测中的作用,我们从ICU选择一批脓毒症病人进行了临床研究,并将PTX3与传统的脓毒症生物标志物降钙素原(PCT)和C-反应蛋白(CRP)进行了比较。结果显示,与SIRS组相比,脓毒症组血清PTX3、CRP和PCT浓度均升高(P0.05)；与脓毒症存活组相比,死亡组的血清PTX3浓度进一步升高(P0.05),而血清CRP和PCT浓度升高没有统计学意义(P0.05)。血清PTX3、CRP、PCT浓度和APACHEII评分的ROC曲线分析显示,与传统的生物标志物CRP.PCT和APACHE Ⅱ评分相比,PTX3是一个更有效的脓毒症诊断和预后预测的生物标志物。PTX3用于脓毒症诊断时的ROC曲线下面积为0.963,当其浓度高于2.43ng/ml时诊断脓毒症的敏感性和特异性均超过90%；PTX3用于脓毒症预后预测时的ROC曲线下面积为0.928,当其浓度高于3.16ng/ml时预测脓毒症预后的敏感性和特异性均超过80%。综上所述,本研究采用iTRAQ标记蛋白质组学方法筛选出一批与大鼠脓毒症诊断和预后预测相关的生物标志物,并选择最佳生物标志物建立了脓毒症诊断和预后预测模型,发现了六个与脓毒症诊断相关的生物标志物：PTX3, MMRN1, FCN1, CPN2, PRSS1和PF4,并发现四个与脓毒症预后预测相关的生物标志物：MMRN1, PPBP, FGa和FGp。经临床研究发现,PTX3作为脓毒症诊断和预后预测的生物标志物,其效果优于传统的生物标志物PCT和CRP。本研究采用蛋白质组学技术,揭示了一批潜在的、新的脓毒症生物标志物,为脓毒症的诊断、疗效判断及预后预测提供了新的实验证据和研究思路。
[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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R459.7

【参考文献】