基于代谢组学“慢性萎缩性胃炎脾胃湿热证”生物学基础的临床研究

【作者】 王亮；

【导师】 李志红；

【作者基本信息】 北京中医药大学 ， 中医内科学（专业学位）， 2016， 硕士

【摘要】 目的：慢性萎缩性胃炎(CAG)是电子胃镜下和胃黏膜病理组织学的一种表现,病程长,易癌变,病理组织学上表现为胃黏膜组织变薄和固有腺体的减少或萎缩,常伴有胃黏膜肠上皮化生和／或不典型增生,是胃癌前病变的一种,其中肠型胃癌通常易在其基础上发生。因此,CAG一直也是消化系统疾病中较为公认的常见病、多发病和难治病。由于其临床表现不具有特征性,一般以上腹部饱胀、食欲不振、胃脘隐痛、消瘦等为主症,电子胃镜这样的侵入性检查,很多病人因本身耐受能力差而拒绝,使CAG难做到“早期诊断、及时治疗”。长期的临床实践中发现,中医治疗CAG确有其独特的疗效,部分病人可以改变其癌前病变状态。脾胃湿热证是临证中脾胃病实证的常见类型,且基础研究较为广泛和深入,但其中医辨证论治的客观依据尚缺乏统一标准,物质基础也尚不明确,无客观化和定量化的实验室指标来实现个体诊疗的规范化和科学化。故本研究应用核磁共振波谱仪对人体血液样品和尿液样品进行检测,得到相应的核磁共振氢谱,对谱图去水峰后采用0.01化学位移分段积分和归一化处理。所得数据输入SIMCA-P软件,采用主成分分析法(PCA)、偏最小二乘-判别分析法(PLS-DA)、响应序列检测(RPT)等手段分析代谢谱图,定性或定量的研究CAG和CAG脾胃湿热证患者的血液和尿液中的代谢产物,试图揭示CAG和CAG脾胃湿热证的生理、病理状态,尝试寻找其生物学标志物,阐释其可能改变的代谢途径或者证候的代谢标志物,为深入探讨中医辨证施治提供一定的研究思路。本论文分为两个部分：第一部分撰写中、西医文献综述,总结关于慢性萎缩性胃炎脾胃湿热证和代谢组学的认识及进展,第一篇总结慢性萎缩性胃炎和脾胃湿热证中医学研究现状；第二篇阐述代谢组学的优势、研究方法及其与疾病、证候的相关研究进展。第二部分为CAG和CAG脾胃湿热证的代谢组学实验研究,并对结果进行分析和讨论,寻找可能的生物学标志物,试图揭示CAG和CAG脾胃湿热证的生理、病理状态,尝试通过生物学标志物阐释其可能改变的代谢途径。方法：1收集65例慢性萎缩性胃炎患者和40例健康对照组志愿者的血液和尿液样品,运用1H-NMR代谢组学技术得出代谢谱图,归一化处理后把得到的积分数值进行主成分分析法(PCA)得到主成分(PC),再利用PC对各组样品代谢谱图进行偏最小二乘—判别分析法(PLS-DA、正交偏最小二乘-判别分析法(OPLS-DA)和响应序列检测(RPT)等分析、检验手段得到相应结果图,从而直观地显示出各组样品代谢物／群信息并找出特征差异代谢物。2把65例患者中脾胃湿热证28例作为实验组,脾胃虚寒证17例作为对照组,采用上述相同的分析方法针对两组血浆进行代谢组学分析,找出差异性代谢物和其变化趋势。结果：1本研究的65例患者中,女性43例,男性22例,最小年龄29岁,最大年龄70岁,平均年龄是52.54±9.54岁；确定伴有肠上皮化生58例,占总病例数的89.23%。2与健康对照组相比较,CAG患者血浆中葡萄糖、丙氨酸、缬氨酸含量升高；谷氨酸、甘氨酸、乳酸、甜菜碱的含量均降低,差异具有统计学意义(P<0.05)。尿液中黄嘌呤、马尿酸、柠檬酸含量升高,肌酐、组胺酸、肌酸、鸟氨酸、磷酸肌酸的含量均降低,差异具有统计学意义(P<0.05)。3 CAG脾胃湿热证患者的血浆代谢组学研究发现：缬氨酸、乳果糖较脾胃虚寒证患者含量均降低,差异具有统计学意义(P<0.05)；异丁酸、甲酸、肌肽较脾胃虚寒证患者含量均升高,差异具有统计学意义(P<0.05)。结论：1本研究慢性萎缩性胃炎患者平均年龄是52.54±9.54岁,这与中国CAG患者平均诊断年龄为中老年人相符合。其中51～60岁病人数较多,与现行流行病学调查结果相一致。2葡萄糖、丙氨酸、缬氨酸、谷氨酸、甘氨酸、乳酸、甜菜碱、黄嘌呤、马尿酸、柠檬酸、肌酐、组胺酸、肌酸、鸟氨酸、磷酸肌酸等物质参与了CAG的发生发展,有可能成为CAG早期诊断的生物学标志物,进一步说明CAG的发生、发展可能与能量代谢、物质传递、信息传导通路和胃肠道菌群代谢等生化代谢通路紊乱有关。3缬氨酸、乳果糖、异丁酸、甲酸、肌肽差异代谢物可能为区别脾胃湿热证的潜在生物学标志物。更多还原

【Abstract】 Objective:Chronic atrophic gastritis (CAG) is a histopathological manifestation of gastric mucosa under electronic endoscopy, which have longer duration and may be cancerization. The pathogenesis of CAG hasn’t been unified and no effective eradication therapy or interventions have been found. There is no specific clinical manifestations of CAG, and generally performances by upper abdominal fullness, loss of appetite, dull pain in epigastrium, weight loss and etc. These can be the gastric precancerous manifestations and intestinal type of gastric cancer could be developed from CAG. Therefore, CAG is always recognized as the most difficult digestive disease to treat. Many patients refuse to take the examination because the manifestation is faint and the patients couldn’t suffer the invasion of gastrointestinal endoscopes. Thus, it’s hard to achieve "early diagnosis and early treatment" of CAG. In TCM, there are many unique treatments with splendid effects to CAG, which could help some patients improve their precancerous conditions. The development of CAG treatment is limited by the losses of the standards and material basis of its syndrome differentiation and treatment of Spleen-Stomach-Dampness-Heat pattern standard based on TCM theory, and there is no objective and quantitative laboratory index to achieve the standardization and scientization of individual therapy. This study reveals the pathological state, biomarkers, potential pathogenesis and the material basis of Spleen-Stomach-Dampness-Heat pattern of CAG, by using 1H-NMR, LC-MS, GC-MS to qualitative or quantitative detect the blood and urine metabolite of CAG patients, especially the Spleen-Stomach-Dampness-Heat pattern. The results of this study would provide a new idea to study the CAG based on the TCM syndrome differentiation and treatments.This thesis is divided into two parts:The first part is the integrated Chinese and Western medicine literature review, which summarizes the progress of metabolomics used in Spleen-Stomach-Dampness-Heat pattern. The first chapter summarizes the development of CAG Spleen-Stomach-Dampness-Heat pattern research; the second chapter introduces the development and advantages of metabolomics to study the disease and patterns. The second part is experimental results of CAG and Spleen-Stomach-Dampness-Heat pattern based on metabolomics. There are the results and discussion, in this part the results reveal the pathological states of CAG Spleen-Stomach-Dampness-Heat pattern, try to find biomarkers, and find the potential pathogenesis and material basis of this pattern.Methods:(1) We collected blood and urine of 65 CAG subjects and 40 subjects as normal control group, using 1H-NMR metabonomics technology obtained metabolic spectra,after the normalization of the integral values obtained import software principal component analysis (PCA) to give the principal component (PC), re-use PC for each set of samples metabolic spectra partial least squares-discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis France (OPLS-DA), draw the corresponding picture, so as to visually display each set of samples metabolites group information and identify characteristic differences metabolites. (2) In 65 patients, we choose 28 subjects in Spleen-Stomach-Dampness-Heat as the experimental group,17 cases of CAG Stomach Deficiency Syndrome as the control group, using the same analytical methods described above metabolomic analysis of two sets of plasma, to identify all the differences between two substances trends in the group.Results:(1) 65 patients in this study, female 43 cases,22 males and 30 years minimum age, maximum age 70 years, mean age was 52.54±9.54 years; diagnosed with intestinal metaplasia in 58 cases,89.23% of the total number of cases. (2)The results of this experiment found that:compared with the healthy control group, CAG plasma glucose, alanine, valine levels increased; glutamic acid, glycine, lactic acid, betaine, mannitol and other differences in metabolite levels were reduced, the difference was statistically significant (P<0.05). Urine xanthine, hippuric acid, citric acid content increased, creatinine, histidine, creatine, ornithine, creatine phosphate and other differences between Generations content were lower, the difference was statistically significant (P<0.05).(3) Plasma Metabolic Research Group for Traditional Chinese Medicine Spleen found:valine, lactulose compared spleen Deficiency Syndrome levels were lower, the difference was statistically significant (P<0.05); isobutyric acid, formic acid, carnosine relatively empty stomach content were higher in patients with cold syndrome, the difference was statistically significant (P<0.05).Conclusion:(1) Chronic atrophic gastritis patients mean age was 52.54±9.54 years, which is China CAG patients with an average age at diagnosis was little difference in the elderly. Wherein the more the number of 51 to 60 year-old patient, consistent with the existing epidemiological findings.(2) Glucose, alanine, valine, glutamic acid, glycine, lactic acid, betaine, xanthine, hippuric acid, citric acid, creatinine, histidine, creatine, ornithine, creatine phosphate and other substances involved in the the development of CAG, CAG may be used as molecular markers for early diagnosis. CAG further explanation of the development of energy metabolism, mass transfer, signal transduction pathways and gastrointestinal flora metabolism and biochemical pathways disorders. (3) Metabolite valine, lactulose, isobutyric acid, formic acid, carnosine may be the difference between CAG Spleen-Stomach-Dampness-Heat potential markers.更多还原