基于UPLC-MS技术的婴儿巨细胞病毒肝炎湿热内蕴证代谢模式研究

发布时间：2018-03-03 23:10

本文选题：婴儿巨细胞病毒肝炎　切入点：湿热内蕴证　出处：《南京中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：巨细胞病毒(Cytomegalovirus,CMV)属疱疹病毒p亚科,是先天性感染中较常见的病毒。我国是CMV感染的高发地区,80%的小儿在3岁前已感染该病毒。CMV感染临床发病及转归受到机体免疫力影响,在孕妇、婴儿和免疫抑制的个体,可引起严重疾病,如胎儿畸形、婴幼儿CMV肝炎、CMV肺炎、多器官功能损害等。婴儿CMV肝炎是目前国内最常见于婴儿期的肝脏疾病,表现为黄疸消退延迟或逐渐加重,伴肝脾肿大及肝功能异常。肝脏炎症较为严重的患儿出现肝纤维化,进一步造成胆汁淤积。伴见胆道狭窄梗阻或胆道闭锁的患儿预后较差。西医治疗该病以抗病毒药物和对症支持治疗为主,效果不甚理想,且常用抗病毒药物更昔洛韦存在骨髓抑制及肝、肾功能损害等不良反应,限制其临床使用。本课题组长期从事婴儿CMV肝炎的基础和临床研究,对CMV的致病机理、病因病机、证候规律和中药治疗进行了深入研究,但对于该病证型的证候本质尚不清楚。本人所在的南京中医药大学中医儿科学研究所具备较为完善的代谢组学分析平台,故本课题组采用代谢组学这一现代技术手段,结合临床生化检查,对CMV肝炎最常见的湿热内蕴证进行证本质研究。目的：研究CMV肝炎湿热内蕴证患儿血浆及尿液样本的代谢产物,探求CMV肝炎湿热内蕴证的证本质。方法：搜集符合诊断标准的CMV肝炎湿热内蕴证组患儿20例,同时搜集20例正常婴儿设为正常组。分别采集两组的血浆和尿样,采用超高效液相色谱-二维线性离子阱质谱联用仪检测两组样本,利用主成分分析和正交偏最小二乘法进行统计分析,筛选潜在的差异性代谢物,分析差异代谢物的代谢通路,探求CMV肝炎湿热内蕴证的证本质。结果：1.血浆分析结果显示,CMV肝炎湿热内蕴证组和正常组OPLS-DA模型参数分别为R2Y=0.980,Q2=0.869(血浆上层样本)和R2Y=0.950,Q2=0.898(血浆下层样本)。正常组和CMV肝炎湿热内蕴证组沿t[1]轴能完全分开,说明两组的代谢模式存在明显差异。共鉴定18个差异代谢物,其中SM(d18:1/18:0)、TG(16:0/18:1/14:1)、TG(16:0/18:1/18:0)等代谢物在患儿体内呈上调趋势；DG(14:0/0:0/22:2)、TG(15:0/18:4/15:0)在患儿体内呈下调趋势。2.尿液分析结果显示,CMV肝炎湿热内蕴证组和正常组OPLS-DA模型参数分别为R2Y=0.882,Q2=0.835,正常组和CMV肝炎湿热内蕴证组完全分开,说明两组代谢产物差异显著。最终鉴定出8个差异代谢物,其中Norvaline、Alanyl-Leucine等在患儿体内呈上调趋势；Urocanic acid呈下调趋势。结论：1.UPLC-LTQ/Orbitrap-MS技术能够较好区分CMV肝炎湿热内蕴证与正常婴儿的代谢模式,并对CMV肝炎湿热内蕴证的代谢模式进行了初步阐释。2.CMV肝炎湿热内蕴证患儿血浆和尿液主要存在鞘脂代谢、甘油磷脂代谢及组氨酸代谢紊乱。
[Abstract]:Cytomegalovirus (CMV) belongs to the subfamily of herpesvirus p, which is a common virus in congenital infection. In China, 80% of children have been infected with CMV before the age of 3 years, and the clinical pathogenesis and outcome of CMV infection are affected by the immunity of the body. In pregnant women, infants and immunosuppressive individuals, it can cause serious diseases, such as fetal malformation, infantile CMV hepatitis pneumonia, multiple organ dysfunction and so on. Infantile CMV hepatitis is the most common liver disease in China at present. Jaundice was delayed or gradually aggravated, accompanied by hepatosplenomegaly and abnormal liver function. Liver fibrosis occurred in children with severe liver inflammation. The prognosis of children with biliary stricture obstruction or biliary atresia was poor. Western medicine was used to treat the disease mainly with antiviral drugs and symptomatic support. In addition, ganciclovir, a common antiviral drug, has adverse reactions such as bone marrow inhibition, liver and kidney function damage and so on, which restrict its clinical use. Our group has been engaged in the basic and clinical studies of infantile CMV hepatitis for a long time, and has been involved in the pathogenesis, etiology and pathogenesis of CMV. The syndromes and TCM treatment have been studied deeply, but the nature of syndromes of the syndrome type is not clear. The Institute of traditional Chinese Medicine and Pediatrics of Nanjing University of traditional Chinese Medicine has a relatively perfect platform for metabonomics analysis. Therefore, our group adopted the modern technique of metabonomics and combined with clinical biochemical examination. Objective: to study the metabolites of plasma and urine samples in children with CMV hepatitis with dampness and heat accumulation syndrome. To explore the syndromes essence of CMV hepatitis damp-heat accumulation. Methods: 20 cases of CMV hepatitis dampness and heat accumulation syndrome group were collected, and 20 normal infants were collected as normal group. Plasma and urine samples were collected from two groups, respectively. Two groups of samples were detected by ultra-high performance liquid chromatography-two-dimensional linear ion trap mass spectrometry. Principal component analysis (PCA) and orthogonal partial least square method were used for statistical analysis to screen potential differential metabolites and analyze metabolic pathways of differential metabolites. To explore the syndromes of CMV hepatitis dampness and heat accumulation. Results: 1. The results of plasma analysis showed that the parameters of OPLS-DA model of CMV hepatitis with damp-heat accumulation syndrome group and normal group were R2YP0. 980 Q2 0. 869 (plasma upper sample) and R2Y + 0. 950 Q 2 + 0. 898 (plasma substratum sample. Normal group and CMV liver group). The syndrome group of internal accumulation of inflammation, dampness and heat can be separated completely along the axis of t [1]. There were significant differences in metabolic patterns between the two groups. A total of 18 different metabolites were identified. Among them, the metabolites such as SMG18: 1 / 1 / 1 / 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1 / 1: 1: 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 1 / 0: 1: 1 / 1 / 1: 1: 1 / 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1: 1:. The results showed that there were significant differences in metabolites between the two groups. Finally, 8 different metabolites were identified. Among them, Norvaline Alanyl-Leucine showed an upward trend in children and Urocanic acid was down-regulated. Conclusion: 1. UPLC-LTQ / Orbitrap-MS technique can distinguish the damp heat accumulation of CMV hepatitis from the metabolic pattern of normal infants. The metabolic pattern of CMV hepatitis damp-heat accumulation syndrome was preliminarily explained. 2. There were mainly sphingolipid metabolism, glycerol phospholipid metabolism and histidine metabolism disorder in plasma and urine of children with CMV hepatitis damp-heat accumulation syndrome.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R272

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