小猪急性肝衰竭模型的蛋白组学研究及临床验证

发布时间：2018-01-13 09:04

本文关键词：小猪急性肝衰竭模型的蛋白组学研究及临床验证　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：背景肝脏疾病种类繁多,病因复杂,每年因肝脏疾病死亡的人数逐年增加。其中,急性肝衰竭是一种病情发展迅速,死亡率高且治疗手段有限的疾病,早期及时的诊断及准确适宜的治疗手段是改善患者预后的关键。目的本研究通过对实验小猪急性肝衰竭模型的序贯性血样的血清蛋白组学研究,描述疾病过程中蛋白质的动态变化,筛选潜在的生物学标志物,最终转化至临床验证,为急性肝衰竭的早期诊断及预后提供依据。方法选取38头实验小猪静脉给药D-氨基半乳糖(1.3g/kg)建立急性肝衰竭模型。给药前(P0)及给药后36小时(P36)的混合血清(n=10)经利用肽配体库(CPLL)技术处理后分为高丰度表达部分(HAP)及低丰度表达部分(LAP),通过蛋白组非标记定量(LFQ)检测获得差异蛋白列表。另选8头实验小猪动态时间点的血清样品,预处理后经平行反应监测(PRM)靶向定量初筛所得的差异蛋白列表。同时,对实验小猪不同时间点的肝组织进行透视电镜、病理学、聚合酶链式反应(PCR)及免疫组化等综合评估,从基因水平到组织蛋白水平检验最终选择的蛋白标志物。最后,通过酶联免疫吸附测定(ELISA)分别检测急性肝衰竭患者及健康人的血清蛋白标志物(果糖-1,6-二磷酸酶,FBP1;视黄醇结合蛋白4,RBP4)浓度。结果基于数据依赖性采集(DDA)所得的鸟枪法对比检测正常状态及急性肝衰竭状态下的小猪血清(n=10)蛋白质表达,结果共发现1589种血清蛋白质,其中变化显著的差异蛋白325种。继而经过数据非依赖性采集(DIA)检测,结果分析可得可靠蛋白谱图137种,蛋白质生物功能分析提示其中线粒体及能量代谢蛋白变化最为显著,共20种。平行反应监测(PRM)外部检验另8头实验小猪完整时间点的20种能量代谢相关蛋白的动态变化。结果提示除了视黄醇结合蛋白4(RBP4)给药后即急速下降、剩余19种蛋白质均持续上升。同时,透视电镜可见急性肝衰竭状态下的肝细胞线粒体退行性改变,体积明显肿胀、嵴消失、数目明显减少;线粒体特异性基因(细胞色素氧化酶1/2,COX1/2)的聚合酶链式反应结果显示其基因表达量给药后显著减少。最终选择视黄醇结合蛋白4(RBP4)和果糖-1,6-二磷酸酶(FBP1)为候选蛋白标志物。对候选标志物的小猪肝脏免疫组化检验结果与血清蛋白组学结果一致。通过酶联免疫吸附测定(ELISA)检测急性肝衰竭患者血清,可见相比健康人,急性肝衰竭患者血清果糖-1,6-二磷酸酶浓度明显升高(p0.05)、视黄醇结合蛋白4浓度显著降低(p0.001)。根据随访资料对急性肝衰竭患者进行短期(30天)生存分析,发现果糖-1,6-二磷酸酶具有与肝病终末期模型(MELD)评分相当的短期预后价值(曲线下面积AUC=0.778),且血清浓度高的患者(16.89μg/dL)相比浓度低的患者预后较差。结论急性肝衰竭过程中线粒体形态功能均明显受损,能量代谢相关蛋白持续性升高。其中,视黄醇结合蛋白4及果糖-1,6-二磷酸酶可能可以作为急性肝衰竭的早期诊断及预后的生物学标志物。
[Abstract]:The background of liver disease variety, complex etiology, every year the number of liver disease death has increased year by year. Among them, acute liver failure is a rapid progression of the disease, high mortality and limited medical treatment disease, early diagnosis and timely accurate and appropriate treatment is the key to improve the prognosis of the patients. The purpose of this study of serum protein group based on the experimental pig model of acute liver failure by sequential blood studies, describes the dynamic changes of protein in disease process, screening of potential biological markers, eventually transformed into clinical verification, provide the basis for early diagnosis and prognosis of acute liver failure. Methods 38 head pig experimental intravenous administration of D-galactosamine (D- 1.3g/kg) to establish the model of acute liver failure. Before administration (P0) and 36 h after administration (P36) of the mixed serum (n=10) by using peptide ligand library (CPLL) after the treatment were divided into high abundance table As part of (HAP) and low abundance expression (LAP), through the group of non protein labeling (LFQ) detection of differential protein list. Serum samples and 8 experimental pig head dynamic time points, after pretreatment by parallel reaction monitoring (PRM) targeting quantitative screening the differential protein list. At the same time, the liver tissues of experimental pigs at different time points of Pathology, transmission electron microscopy, polymerase chain reaction (PCR) comprehensive evaluation and immunohistochemistry, protein markers from gene level to protein level test the final choice. Finally, by means of enzyme-linked immunosorbent assay (ELISA) were detected in serum of patients with acute liver failure and the health markers (two fructose -1,6- phosphatase, FBP1; retinol binding protein 4, RBP4) concentration. Based on the results of data dependent acquisition (DDA) the detection of shotgun compared with normal state and acute liver failure under the condition of pig Serum protein (n=10) expression, the results of the 1589 serum proteins were found, including the significant changes of 325 kinds of proteins. Then through data dependent acquisition (DIA) detection, result analysis can get reliable protein spectra of 137 kinds of biological function of protein analysis showed that the mitochondrial energy metabolism and protein changes were most significant, a total of 20. Parallel reaction monitoring (PRM) external test of dynamic change of 20 kinds of proteins related to energy metabolism on the other 8 experimental pigs complete time points. Results suggest that in addition to retinol binding protein 4 (RBP4) after administration is rapidly decreased, the remaining 19 proteins have continued to rise. At the same time, the perspective of hepatocyte mitochondria degeneration electron microscope of acute liver failure under the condition of the change of volume was swelling, cristae disappeared, significantly reduce the number of mitochondria; specific gene (cytochrome 1/2, COX1/2) the results showed that the polymerase chain reaction Gene expression was significantly reduced after administration. The final selection of retinol binding protein 4 (RBP4) and two -1,6- fructose phosphatase (FBP1) as candidate protein markers. The pig liver immune group of candidate markers of inspection results and serum proteomic results. The determination of absorption by enzyme linked immunosorbent assay (ELISA) detection of serum patients with acute liver failure, compared to healthy people, serum -1,6- in patients with acute liver failure two fructose phosphatase concentration increased significantly (P0.05), retinol binding protein 4 concentrations were significantly decreased (p0.001). According to the short-term follow-up data on acute liver failure patients (30 days) survival analysis, found that fructose -1,6- two with liver phosphatase end at the end of the model (MELD) score of the short-term prognostic value of equivalent (area under the curve AUC=0.778), and serum concentration in patients with high (16.89 g/dL) compared with the low concentration of poor prognosis. Conclusion acute liver failure process Mitochondrial morphology and function were significantly impaired, and energy metabolism related proteins continued to rise. Among them, retinol binding protein 4 and fructose -1,6- two phosphatase could be used as biomarkers for early diagnosis and prognosis of acute liver failure.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R575.3;R-332

【相似文献】