慢性乙型肝炎组织学肝硬化血清标志物筛查的研究

发布时间：2018-04-05 02:09

本文选题：慢性乙型肝炎　切入点：组织学肝硬化　出处：《大连医科大学》2015年硕士论文

【摘要】：研究背景及目的:肝纤维化是由致病因子引起的肝脏内结缔组织异常增生,肝内细胞外基质弥漫性沉积过度的病理过程。肝纤维化的形成是由于机体感染病毒时,引起肝脏炎症反应的同时,激活了肝脏的免疫系统修复组织,修复的过程过度或者失控时,导致肝功能异常和肝脏结构的改变。轻者为肝纤维化,严重者造成肝小叶的结构发生紊乱,逐步形成假小叶,最终演变为肝硬化。大量的临床研究结果表明[1],人体自身内部存在纤维降解系统,该系统可以使肝纤维化实现逆转,甚至早期的肝硬化也可能发生逆转[2][3],而肝组织损坏严重的失代偿期肝硬化则不能逆转。因此,早期诊断和治疗肝纤维化,就可以阻止肝纤维化向肝硬化的方向进展,这对于改善慢性肝病患者的预后有着极其重要的意义。早期的肝纤维化的血清标志物主要反映肝脏胶原的合成和分解代谢的动态变化,不能真正反映胶原蛋白的沉积程度。目前公认的肝纤维化诊断“金标准”是肝脏穿刺病理学检查。由于该检查具有创伤性及局限性,并且患者要忍受一定的痛苦,重复检查的可能性较小,因此其临床应用受到限制。影像学检查对重度肝纤维化诊断比较可靠,而对轻、中度肝纤维化的诊断则缺乏特异性。目前临床缺乏评价肝纤维化程度[4]的实用的血清标志物,所以,找出早期诊断肝纤维化、评价肝纤维化程度的血清标志物来指导临床的诊治是当前医学的一项重要研究课题。代谢组学(metabonomics/metabolomics)是主要研究生物被扰动后所产生代谢产物的种类、数量以及其中变化规律的一门科学。代谢组学已被应用到肝衰竭[5]、非酒精性脂肪肝[6]、原发性肝癌[7][8]、药物和毒物等非病毒性因素引起的肝脏损伤[9][10]等的早期标志物标志物筛查中,为疾病的早期诊断和治疗提供了新的方法。本实验采用高效液相色谱-轨道离子阱质谱联用质(uplc/ltq-orbitrapms)技术来分离并提取慢性乙型肝炎、代偿期肝硬化以及失代偿期肝硬化患者血清中的代谢产物,以期发现肝纤维化的代谢产物标志物群。方法:1.随机选取2011年10月-2013年9月于烟台市传染病医院就诊的慢性乙型肝炎患者34例,所有患者均接受了肝穿刺病理检查,随机选取于大连市第六人民医院住院治疗的失代偿期肝硬化患者15例。同期选择20例健康志愿者作为正常对照组,肝功正常、病原学检查阴性。所有入组人员在知情同意的前提下,留取空腹血4ml,半小时内将血清分离,留取血清,-80℃冰箱冷冻保存备检。2.高效液相色谱-轨道离子阱质谱联用质谱(uplc/ltq-orbitrapms)技术检测所有患者血清中的代谢产物。2.1样品准备:室温下静置解冻血清,取100ul血清加400ul甲醇除蛋白,涡旋60秒,13000rpm离心15分钟,取上清液400ul置冷冻浓缩仪冻干。2.2超高效液相色谱分析:采用超高效液相色谱分析仪。冲洗柱采用10cm×2.1mm×1.7umc18柱根据梯度不同而改变的乙腈溶液冲洗,根据物质在柱内停留时间的长短,完成各种代谢产物的分离。2.3高效液相色谱-轨道离子阱质谱联用质谱(uplc/ltq-orbitrapms):通过采用ltq-orbitrap质谱分析仪,对已经分离的代谢产物实行esi+模式操作。2.4处理数据和统计学分析:通过micromassmarkerlynx获取每峰停留时间(tr)和m/z数据对,标准化输出每峰离子密度后,进行主成分分析(pca)。根据非参检验结果,寻找差异比较大的代谢产物。spss.18进行统计学分析。2.5确定代谢产物名称:根据精确分子量来分析、研究确定代谢产物名称。结果:1.对慢性乙型肝炎34例进行肝脏穿刺进行病理检查,结果显示:s1:8例,s2:5例,s3:1例,s4:20例。其中s1-s3期14例,为慢性乙型肝炎组;s4期20例,为早期肝硬化组即代偿期肝硬化组。2.在对正常对照组、慢性乙型肝炎组、代偿期肝硬化组、失代偿期肝硬化组患者血清物质代谢谱的比较中发现,相对正常对照组,慢性乙型肝炎、代偿期肝硬化、失代偿期肝硬化患者蛋氨酸呈逐渐下降趋势,辅酶Q1、羧乙基精氨酸、甘氨脱氧胆酸呈逐渐上升趋势。结论:蛋氨酸(Methionine)、辅酶Q1(coenzyme Q1)、羧乙基精氨酸(octopine)、甘氨脱氧胆酸(GDCA)可作为肝纤维化的代谢标志物群。
[Abstract]:Background and objective: hepatic fibrosis is abnormal hyperplasia of connective tissue caused by pathogenic factor in the liver, the deposition of the pathological process of excessive extracellular matrix diffuse liver. Hepatic fibrosis is due to infection when the virus causes inflammation of the liver and activate the immune system of the liver tissue repair, the repair process of excessive or out of control, leading to abnormal liver function and liver structure changes. The light of hepatic fibrosis, hepatic lobule structure causing serious disorder, gradually formed pseudolobule, eventually evolved into cirrhosis. Results many clinical studies showed that [1], fiber degradation system of the human body itself exists, the system can make the realization of hepatic fibrosis reversal even early cirrhosis may also be reversed [2][3], and liver tissue damage serious decompensated hepatic cirrhosis is irreversible. Therefore, early diagnosis and treatment Hepatic fibrosis, can prevent the progress of liver fibrosis to cirrhosis, which is of great significance to improve the prognosis of patients with chronic liver disease. The serum markers of liver fibrosis in the early stage mainly reflects the dynamic changes of liver collagen synthesis and catabolism, can not truly reflect the degree of collagen deposition. The hepatic fibrosis diagnosis "recognized the gold standard is the pathological examination. The examination is traumatic and limitations, and patients suffer some pain, less likely to repeat the inspection, so its clinical application is restricted. Imaging examination in the diagnosis of severe liver fibrosis is reliable, and the light, moderate liver fibrosis is the lack of diagnosis the lack of specificity. The clinical utility of serum markers to evaluate the degree of liver fibrosis, [4] so, find out the early diagnosis of liver fibrosis, liver fibrosis evaluation The level of serum markers to guide the clinical diagnosis and treatment is an important subject in current medicine. Metabonomics (metabonomics/metabolomics) is a kind of metabolites produced by biological research after disturbance, a number of science and which changes in the law. Metabonomics has been applied to non alcoholic liver failure [5] fatty liver [6], hepatocellular carcinoma [7][8], caused by drugs and poisons and other non viral factors of liver injury [9][10] markers for early screening biomarkers, which provides a new method for early diagnosis and treatment of diseases. This experiment using high performance liquid chromatography - mass ion trap mass spectrometry (mass uplc/ltq-orbitrapms) technology to separate and extract the chronic hepatitis B, cirrhosis and decompensated serum metabolites in patients with liver cirrhosis, in order to find the metabolites of liver fibrosis markers. Method: 34 patients with chronic hepatitis B were randomly selected from the 1. October 2011 -2013 year in September to visit Yantai city hospital for infectious disease patients, all patients underwent liver biopsy, randomly selected from the Sixth People's Hospital of Dalian city hospital treatment of patients with decompensated liver cirrhosis in 15 cases. In the same period 20 cases of healthy volunteers as normal control group, normal liver function, pathogenic examination negative. All groups in the premise of informed consent, collected fasting blood 4ml, serum separation within half an hour, the serum -80, C refrigerator cryopreservation inspection equipment.2. high performance liquid chromatography ion trap mass spectrometry with orbit (uplc/ltq-orbitrapms) technique for the detection of metabolites all samples of.2.1 in the serum of patients with preparation: room temperature static thawing serum 100ul, serum 400ul and methanol deproteinization, vortex 13000rpm centrifugal 60 seconds, 15 minutes, the supernatant was 400ul the frozen freeze concentration instrument .2.2 ultra high performance liquid chromatography using ultra performance liquid chromatography analyzer. Rinse the acetonitrile solution column using 10cm * 2.1mm * 1.7umc18 column is changed according to the different gradient washing, according to the residence time of material in the column length, to complete a variety of metabolites of.2.3 separation by high performance liquid chromatography ion trap mass spectrometry track mass spectrometry (uplc/ltq-orbitrapms) by using ltq-orbitrap mass spectrometry analysis and statistical data, the operation of the.2.4 processing mode of esi+ metabolites have been isolated: obtaining each peak retention time by micromassmarkerlynx (TR) and m/z data to standard output, each peak ion density, principal component analysis (PCA) based on non parametric test. The search for the difference of metabolic products of.Spss.18 were analyzed to determine the.2.5 metabolic product name: according to the accurate molecular weight analysis, determination of metabolites Name. Results: 1. of 34 cases of chronic hepatitis B liver puncture biopsy, the results showed that: s1:8 cases, s2:5 cases, s3:1 cases, s4:20 cases. 14 cases of S1-S3 patients and chronic hepatitis B group; 20 cases of stage S4, early cirrhosis group decompensated cirrhosis group.2. in normal control group, chronic hepatitis group, cirrhosis group, found in decompensated cirrhosis group serum substance in patients with metabolic spectrum comparison, relative to the normal control group, chronic hepatitis B, cirrhosis, decompensated cirrhosis patients with loss of methionine decreased gradually, coenzyme Q1, octopine, glycodeoxycholate increased. Conclusion: methionine (Methionine), coenzyme Q1 (coenzyme Q1), carboxyethyl arginine (octopine) and glycodeoxycholate (GDCA) can be used as a metabolic marker of liver fibrosis group.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R512.62;R575.2

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