实验室检验指标用于协助决策早产NEC患儿手术的价值和血清定量差异蛋白组学分析

发布时间：2018-07-08 11:52

本文选题：早产儿 + 坏死性小肠结肠炎　；参考：《大连医科大学》2017年硕士论文

【摘要】：第一部分实验室检验指标用于协助决策早产NEC患儿手术的价值背景NEC(necrotizing enterocolitis,坏死性小肠结肠炎)是早产儿急性肠道炎症性疾病,病死率高,预后相对较差。临床可表现为胃潴留、腹胀、便血等肠道症状,又可表现为呼吸暂停、反应低下、休克等全身中毒症状。目前临床医生对此疾病的治疗原则和方法已相对统一,包括绝对禁食、持续胃肠减压、抗生素的应用等内科保守治疗方法和坏死肠管切除等外科手术治疗。ⅡB期至ⅢA期之间的NEC患儿,疾病发展变化快,表现又无明显特殊性。此时手术的相对适应症难以把握,这时应用内科保守治疗能否控制病情的发展,还是需积极手术去除肠道病灶,是否有临床特异性的指标来协助治疗,是我们亟需思考的问题。目的本文将讨论早产NEC患儿发病后前三天不同实验室检验指标与NEC手术的相关性,选择可信度更高的指标评估患儿NEC手术的需要,以更好把握临床手术时机。方法本研究收集2014年12月至2015年7月我院极早产NICU科室收住的30例胎龄33周、Bell分期Ⅱ期以上的NEC患儿的临床资料。根据治疗方案分为手术组16例,未行手术治疗的保守组14例。将早产NEC手术治疗组和保守组发病后连续三天内实验室检验指标,应用SPSS 19.0统计软件进行分析比较。研究因素包括:发病后三天内白细胞计数、粒细胞比值、血小板计数、C反应蛋白数值。结果在研究因素中,早产NEC患儿发病第一天的白细胞计数和CRP值,第二天的血小板计数和CRP值,第三天的血小板计数和CRP值与NEC手术相关。发病后第二天的血小板计数的减低是NEC手术的独立高危因素。针对发病后第二天血小板计数进行ROC曲线分析计算出cut-off值为144.5*109/l,对应的敏感性为81.3%,特异性为87.5%,阳性预测值为89.45%,阴性预测值为91.52%。结论1.早产NEC患儿发病第一天的白细胞计数和CRP值,第二天的血小板计数和CRP值,第三天的血小板计数和CRP值与NEC手术治疗相关。2.发病后24-48小时内血小板计数是NEC手术的独立高危因素。当数值低于144.5*109/l时,提示手术需要性高,我们需要结合患儿临床进程情况,决策更好的治疗方案,把握最佳手术时机。第二部分早产NEC患儿血清定量蛋白组学分析背景NEC可导致肠道急性炎症性病变,其发病原因错综复杂,更容易侵袭肠道发育不成熟、功能低下以及免疫功能弱的早产新生儿。此病无特异性表现及检验标准,进展快,疾病预后差,对新生儿生长发育和生活质量影响极大。现多认为NEC的发病机理为肠上皮屏障功能低下或损伤、肠道血供调节差及饮食的影响,大量条件致病菌滋生或菌群易位等等。从而大量炎症介质产生,引起肠上皮细胞损伤,肠壁坏死穿孔、全身炎症反应综合征甚至休克、多器官衰竭。虽然近年有关NEC的研究热度持续升高,但其相关的分子机制的组学文献亦较少。差异蛋白组学是分析不同时期或不同状态下,动态变化的蛋白质组之间的差异。TMT(Tandem Mass Tags,串联质谱标签)技术是应用同位素标签,特异性标记多肽氨基集团,进行质谱分析,并行计量了多样品中蛋白质的相对含量。目的通过TMT技术研究早产NEC患儿血清定量蛋白组学,利用分子细胞学信息,获取对疾病发生发展机制更深层的认识。方法:收集2014年12月在我院极早产NICU住院治疗双胎1对,其中1例为明确诊断NEC患儿,Bell分期为ⅢA,生后24小时入院,胎龄为30周,NEC发生前喂养量达110ml/kg.d。另1例为与NEC患儿同期住院的双胞胎(没有罹患NEC、病情相对稳定、没有严重感染)。将血样收集,血清去高丰度蛋白,经过样品质量检验、TMT标记全蛋白、C18色谱柱样品分级、质谱相对定量检测和质谱数据分析得出差异蛋白。将所有差异蛋白的基因ID信息导入KOBAS 2.0软件,进行信号通路、疾病和GO富集分析。结果:1.共鉴定和定量了1515种蛋白。选择表达上调2倍和表达下调0.5,且配对t检验p值小于0.05,作为定义差异蛋白的筛选标准,共发现44种差异蛋白,其中表达显著上调的有24种,下调的有20种。2.显著富集的通路包括血小板活化、补体和凝血级联反应、蛋白降解、活性氧类物质解毒、外源性抗原交叉呈递、碳水化合物代谢、细胞调亡的调节。显著富集的信号通路涉及到TGF-β受体信号传导通路、P53信号通路、Hedgehog信号通路等等。结论:1.TMT定量蛋白质组学技术可有效用于筛选NEC患儿差异表达的蛋白质;2.本研究通过对NEC患儿特异血清标记物和发病机制进行初步探索,筛选出可信度高的44种差异蛋白质,显著富集的生物过程包括血小板活化、活性氧类物质解毒、伤口愈合等,多种蛋白也与参与到血小板脱颗粒、血小板活化等生物活性中,结合第一部分内容,揭示了在多种病理因素作用下,血小板相关生物过程与NEC的发生有直接的关系;3.本次研究在蛋白组学的基础筛选出显著富集的信号通路,提示NEC疾病的发生涉及了TGF-β受体信号传导通路、P53信号通路、Hh信号通路等等,它们在病程中介导了上皮间质转化、肠道细胞调亡、肠道组织损伤和修复、维持肠道紧密连接屏障完整性等生物过程。
[Abstract]:The first part of the laboratory test index is used to assist in the operation of children with premature delivery of NEC. The value background NEC (necrotizing enterocolitis, necrotizing enterocolitis) is an acute intestinal inflammatory disease in preterm infants with high mortality and relatively poor prognosis. The treatment principles and methods of the disease have been relatively unified, including the absolute fasting, continuous gastrointestinal decompression, the application of antibiotics and the surgical treatment of necrotic bowel resection, and so on. Second, NEC children between phase II B to stage III A, rapid development of disease, table There is no obvious specificity at the moment. At this time, the relative indications of the operation are difficult to grasp. At this time, it is a problem we need to think about whether conservative treatment can control the development of the disease, whether it needs active operation to remove the intestinal focus, and whether there are clinical specific indicators to help the treatment. The purpose of this article will be to discuss the first three of the preterm NEC children. The correlation between the test indexes of different laboratory and the NEC operation, and choosing a more reliable index to evaluate the needs of NEC operation in children to better grasp the time of clinical operation. Methods this study collected the clinical data of 30 children aged 33 weeks of fetal age and more than NEC in Bell staging period from the NICU Department of our hospital from December 2014 to July 2015. According to the treatment scheme, 16 cases of the operation group and 14 cases of the conservative group were treated without operation. The laboratory test indexes of the premature NEC operation group and the conservative group within three days after the onset of the disease were analyzed and compared with the SPSS 19 statistical software. The factors included the white cell count, the granulocyte ratio, the platelet count, the C reaction within three days after the onset of the disease. Results in the study, results in the study factors, the first day of the onset of NEC children's leukocyte count and CRP value, second days of platelet count and CRP, third days of platelet count and CRP value associated with NEC surgery. Second days after the onset of platelet count is an independent risk factor for NEC operation. Second days after the onset of the disease, the blood is small. The ROC curve analysis showed that the cut-off value was 144.5*109/l, the corresponding sensitivity was 81.3%, the specificity was 87.5%, the positive predictive value was 89.45%, the negative predictive value was 91.52%. conclusion 1. preterm NEC children's leukocyte count and CRP value, second day blood plate count and CRP value, third day platelet count and CRP value. Platelet count is an independent risk factor for NEC surgery within 24-48 hours after the onset of.2. associated with NEC operation. When the value is lower than 144.5*109/l, it is suggested that the operation needs to be high. We need to combine the clinical process of the children, make a better treatment plan, grasp the best operating time machine. Second part of the serum quantitative protein group of premature NEC children. Background NEC can lead to acute inflammatory bowel disease. The cause of the disease is complicated, it is more likely to attack premature infants with immature intestinal development, hypofunction and weak immune function. The disease has no specific performance and test criteria, rapid progress, poor prognosis, and great influence on the growth and quality of life of the newborn. The pathogenesis of NEC is the hypofunction and damage of the intestinal epithelial barrier, the poor regulation of the intestinal blood supply and the influence of diet, a large number of pathogenic bacteria breeding or bacterial translocation, etc., resulting in a large number of inflammatory mediators, causing intestinal epithelial cell damage, intestinal wall necrosis and perforation, systemic inflammatory reaction syndrome even shock, and multiple organ failure. Although in recent years, there are many organs failure. The research heat of NEC is rising, but the related molecular mechanism is also less. Differential proteomics is the difference of.TMT (Tandem Mass Tags, tandem mass spectrometry label) using the identity label, the specific polypeptide amino group, and the analysis of the difference between different periods or different states. The relative content of protein in multiple samples was measured by mass spectrometry. Objective to study the serum quantitative proteomics of children with premature NEC by TMT technology and use molecular cytology information to obtain a deeper understanding of the mechanism of disease development. Methods: 1 pairs of twins in the hospital of NICU in our hospital in December 2014 were collected and 1 of them were identified. The Bell stage was III A, 24 hours after birth, 24 hours after birth and 30 weeks of fetal age. 1 twins were fed before NEC, and 1 were twins hospitalized in the same period of NEC children (no NEC, relatively stable, no severe infection). Blood samples were collected, serum high abundance protein, sample quality test, TMT labelled total protein, C18 The difference protein was obtained by the chromatographic column sample classification, mass spectrometry and mass spectrometric analysis. The ID information of all the differential proteins was introduced into KOBAS 2 software to carry out the signal pathway, disease and GO enrichment analysis. Results: 1., 1515 proteins were identified and quantified. The selection expression was up 2 times and the expression was down 0.5, and the p value of paired t test was small. 0.05, as the screening criteria for defining differential proteins, there were 44 different proteins, of which 24 were significantly up-regulated, and 20 kinds of.2. significantly enriched pathways included platelet activation, complement and coagulation cascade, protein degradation, reactive oxygen species detoxification, exogenous antigen cross presentation, carbohydrate metabolism, cell modulation. TGF- beta receptor signaling pathway, P53 signaling pathway, Hedgehog signaling pathway and so on. Conclusion: 1.TMT quantitative proteomics technology can be used to screen differentially expressed proteins in children with NEC; 2. this study was conducted by preliminary exploration of specific serum markers and pathogenesis of children with NEC. 44 kinds of differential proteins with high reliability were screened out. The significant enrichment of biological processes included platelet activation, detoxification of reactive oxygen species, wound healing and so on. Many proteins also combined with the biological activity of platelet degranulation and platelet activation, combined with the first part of internal volume, and revealed the platelet correlation under the action of various pathological factors. There is a direct relationship between biological processes and the occurrence of NEC; 3. this study screened a significant signaling pathway on the basis of proteomics, suggesting that the occurrence of NEC diseases involves the TGF- beta signaling pathway, the P53 signaling pathway, the Hh signaling pathway and so on, which lead to epithelial mesenchymal transition, intestinal cell apoptosis and intestinal tissue in the course of the disease. Injury and repair, maintain the integrity of intestinal tight junctions and other biological processes.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R722.6

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