维医异常胆液质证与溃疡性结肠炎大鼠模型的组学研究

发布时间：2018-08-09 11:09

【摘要】：目的:本研究论文主要选择维医异常胆液质证模型大鼠与TNBS-乙醇灌肠液诱导的溃疡性结肠炎模型大鼠为研究对象,通过分析大鼠一般体征、下丘脑及结肠组织形态学结构变化;通过代谢组学分析下丘脑、结肠代谢组份差异;通过RNA-Seq测序技术研究下丘脑、结肠转录谱差异;通过研究甲状腺激素异常对异常胆液质证与UC大鼠代谢紊乱产生的作用,综合分析异常胆液质证形成及其诱发UC的物质基础。方法:1)采用干热饲养环境、干热属性饲料(黑胡椒、干姜、西红花)、夹尾、噪音等多因素复合刺激,建立异常胆液质证载体动物模型。通过TNBS乙醇液灌肠复制大鼠溃疡性结肠炎模型,观察大鼠生物表型改变及组织形态学改变;2)利用核磁共振技术检测多因素复合诱导的异常胆液质证、UC大鼠模型中下丘脑和结肠代谢产物的变化;3)应用RNA-seq全面分析异常胆液质证和UC大鼠下丘脑和结肠组织的转录本,筛选出差异表达基因。第二,应用GO方法进行差异基因的功能聚类,应用KEGG信号通路富集差异表达基因相关的信号通路。第三,构建蛋白质相互作用网络,分析差异表达基因之间的相互作用,筛选出重要的差异表达基因。第四,应用实时荧光定量PCR方法验证差异基因的表达水平;4)检测血清中甲状腺功能7项指标及血清糖脂代谢相关生化指标,通过相关性分析,分析甲状腺激素水平与异常胆液质证及溃疡性结肠炎形成中对代谢的影响。同时通过血清代谢组学的结果绘制代谢的整体网络调控图。结果:1)通过多因素复合造模方法成功模拟出了符合人体异常胆液质证临床表现的证候模型以及成功复制了TNBS-UC模型。肉眼观察异常胆液质证结肠组织出现轻微充血,HE染色后镜下观察,发现异常胆液质证组出现少量炎细胞浸润。异常胆液质证组下丘脑镜下出现脑血管扩张充血,血管周围间隙增宽,部分神经细胞形态变圆。UC组结肠肉眼可见肠壁坏死充血水肿现象,部分深大溃疡表面有黑褐色膜状物附着,周边肠黏膜充血、水肿明显,病变部位肠壁明显增厚,管壁僵硬。HE染色后镜下可见黏膜上皮细胞变性坏死,黏膜固有层,黏膜下层大量中性粒细胞浸润伴纤维素渗出及片状出血,符合急性炎症的组织病理学改变,与人类UC急性期改变相似。UC模型组下丘脑镜下出现脑血管扩张充血,血管周围间隙增宽,部分神经细胞形态变圆;2)下丘脑代谢组学研究结果显示异常胆液质证组显著性高峰度代谢物为缬氨酸、亮氨酸、异亮氨酸、乙醇、γ-氨基丁酸、N-乙酰天冬氨酸、胆碱和肌醇,显著性低峰度代谢物为谷氨酸和牛磺酸。UC组具有显著性高峰度代谢物为:乙醇,γ-氨基丁酸,N-乙酰天冬氨酸,乳酸,苯丙氨酸。通过对样本间进行RNA-Seq分析,发现异常胆液质证大鼠在下丘脑中大量的通路与神经信号通路相关,如“Neuroactive ligand-receptor interaction”,“Cocaine addiction”,“Amphetamine addiction”,“Serotonergic synapse”,“dopaminergic synapse”等。UC组在丘脑组织中上调表达的基因富集到“Adipocytokine signaling pathway”、“Calcium signaling pathway”、“Tyrosine metabolism”、“Cytokine-cytokine receptor interaction”、“PI3K-Akt signaling pathway”等通路。异常胆液质证组和UC组差异基因共表达分析,发现2条和甲状腺激素应答相关通路,分别是“Thyroid hormone synthesis”、“Autoimmune thyroid disease”,差异表达基因为Cga,Tshb。结合GO-蛋白网络图和基因通路网络图,找到四个共同上调基因,分别是Pmch、Cga、Tshb和Hcrt;3)结肠代谢组学分析显示异常胆液质证组具有显著性高峰度代谢物为:3-羟基丁酸,乳酸,琥珀酸,ATP/ADP。异常胆液质证组具有显著性低峰度代谢物为:GSH,甘油磷酸胆碱,肌醇。UC组的差异代谢物结果显示,和正常组对比,UC组结肠具有显著性高峰度代谢物为:丁酸,丙酸,乙醇,3-羟基丁酸,乙酸,琥珀酸,甲胺,二甲胺,丙二酸,葡萄糖,糖原。UC组具有显著性低峰度代谢物为:丙氨酸,谷氨酰胺,谷胱甘肽,甘油磷酸胆碱,肌醇,磷酸肌酸,牛磺酸,肌酐。结肠组织获得差异表达基因通过GO和KEGG分析,发现在异常胆液质证组结肠出现了炎症、免疫相关通路基因表达下调,而UC组炎症、免疫相关通路基因表达上调的现象。将异常胆液质证与UC组取并集,运用WGCNA进行分析,选择在正常组和异常胆液质证组低表达,UC组高表达的module进行KEGG通路分析,得到的大部分功能通路与免疫相关,如“Cytokine-cytokine receptor interaction”,“TNF signaling pathway”,“PI3K-Akt signaling pathway”,“NF-kappa B signaling pathway”等信号通路,同时也发现2条甲状腺激素相关的通路,筛选甲状腺激素主要信号通路,通过基因间的相互作用网路关系图,发现Tnf是其中的核心分子。通过Thrsp基因做共表达分析,发现糖异生关键酶PCK1、FBP的下调和GK上调。同时糖酵解途径中PKLR和GPD1出现下调,脂肪酸β-氧化中脂酰Co A脱氢酶(Acadsb)基因表达却上调,谷胱甘肽S转移酶和谷氨酸-半胱氨酸连接酶均下调;4)血清中T3、T4、FT3、FT4水平在异常胆液质证组和UC组均降低,异常胆液质证组虽然降低,但仍然处在正常范围内,UC组明显低于正常水平。血糖、果糖胺、甘油三酯、载脂蛋白B在UC组中显著升高,高密度脂蛋白显著降低,(P0.05)。甲状腺激素与葡萄糖、甘油三酯、总胆固醇存在负相关,与高密度胆固醇正相关。异常胆液质证组最终确定大鼠血清中有13种的差异性代谢成分,其中异常胆液质证组中显著升高的代谢物为:异亮氨酸、丙氨酸、缬氨酸、脯氨酸、丙酮酸、肉碱、β-羟丁酸、乳酸、肌酸,显著降低的代谢物为:VLDL、瓜氨酸、酪氨酸。和正常组比较后,UC组中共找到13种差异代谢物,在UC组中显著升高的代谢物为:LDL、乳酸、肌酸,UC组中显著降低的代谢物为:亮氨酸、缬氨酸、瓜氨酸、酪氨酸、丙氨酸、肉碱、柠檬酸、β-葡萄糖、α-葡萄糖、甲基组氨酸。结论:1)通过干热属性饲料,干热环境喂养,夹尾、噪音、慢性足底电刺激多因素复合方法造模,大鼠出现了出现情绪易激动、喜打斗、舌苔发黄、尿色黄、粪便干硬、体重减轻、饮水、饮食量增加的现象。成功模拟出了符合人体异常胆液质证临床表现的证候模型。成功复制TNBS乙醇液灌肠诱导大鼠溃疡性炎症的发生。模型中结肠组织出现了与人类UC急性期相似的病理改变;2)下丘脑神经递质分泌异常,膜稳定性破坏及能量代谢异常是异常胆液质证组与UC组中的共同表现。TSH编码基因Cga和Tshb表达上调,可能是异常胆液质证组和UC组下丘脑对刺激的应答反应,在异常胆液质证和UC形成过成中都起到非常关键的作用,推测在异常胆液质证诱发UC过程中发挥重要作用;3)结肠组织出现的能量代谢失衡、氧化抗氧化体系平衡紊乱、细胞膜稳定性破坏是导致溃疡性结肠炎发生的主要因素,同时也是异常胆液质证诱发UC的主要原因。TNF-α一方面通过抑制GSH活性使结肠细胞产生氧化损伤,另一方面做为促炎细胞因子,通过激活和放大的双重作用,以DAG-PKC-NF-κB信号传导途径激活而产生炎症反应,在UC形成中起重要作用;4)血清甲状腺激素含量降低是异常胆液质证组与UC组发生的共同物质基础。异常胆液质证与UC形成过程中,神经内分泌系统和免疫系统相互作用,一方面免疫系统通过细胞因子(TNF)作用于神经-内分泌系统,使神经-内分泌系统激素释放异常(甲状腺激素含量降低),另一方面甲状腺激素又作用于全身,使整体代谢发生改变如抑制糖代谢,促进脂类分解,使能量失衡,并产生全身免疫紊乱使结肠受到损伤。异常胆液质证组血清甲状腺激素含量偏低但仍处于正常范围,是异常胆液质证处于亚健康状态的主要原因,也是异常胆液质证诱发UC的原因之一。
[Abstract]:Objective: in this study, the main objective of this study was to select the rat model of ulcerative colitis induced by TNBS- alcohol enema and the rat model of alcohol enema. Through the analysis of the general signs, the morphological changes in the hypothalamus and colon, the difference of the hypothalamus and the metabolism of the colon was analyzed by the metabolic group; RNA-Seq The difference in the hypothalamus and colon transcriptional spectrum of the hypothalamus was studied by sequencing. Through the study of the effect of abnormal thyroid hormone on abnormal bile fluid syndrome and the metabolic disorder of UC rats, the formation of abnormal bile duct syndrome and the material basis of inducing UC were synthetically analyzed. Methods: 1) dry heat feeding environment, dry heat attribute feed (black pepper, dried ginger, crocus), and tail clip, The animal model of abnormal bile liquid syndrome was established by multiple factors such as noise and other factors. The rat model of ulcerative colitis was replicated by TNBS ethanol liquid enema. The changes of biological phenotypes and histomorphology in rats were observed. 2) the abnormal bile fluid syndrome induced by multiple factors was detected by MRI, and the hypothalamus and nodal of UC rat model were found. Changes in intestinal metabolites; 3) using RNA-seq to comprehensively analyze the abnormal bile fluid syndrome and the transcriptional transcript of the hypothalamus and colon of UC rats, select the differentially expressed genes. Second, use the GO method to carry out the functional clustering of the differential genes, and use the KEGG signaling pathway to enrich the signal pathways related to the differential expression basis. Third, the construction of protein each other. Function network, analysis the interaction between differentially expressed genes, screening out important differentially expressed genes. Fourth, using real time fluorescence quantitative PCR method to verify the expression level of differential genes; 4) detection of 7 indexes of thyroid function and biochemical indexes of serum glycolipid metabolism in serum, and analysis of thyroid hormone water through correlation analysis. The effect of flat and abnormal bile fluid syndrome and the formation of ulcerative colitis in formation of ulcerative colitis. At the same time, the overall network regulation of metabolism was plotted through the results of serum metabolomics. Results: 1) the syndrome model and the successful replication of the TNBS-UC model were successfully simulated by multi factor compound modeling method. A small amount of hyperemia in the colon tissue of abnormal bile fluid was observed by the naked eye. After HE staining, a small amount of inflammatory cell infiltration was found in the abnormal bile liquid syndrome group. The abnormal bile fluid syndrome group had dilatation and congestion of the cerebral vessels under the hypothalamic microscope, the widening of the perivascular space and the necrosis of the colon in the.UC group of the group of nerve cells visible to the intestinal wall necrosis. The phenomenon of hyperemia and edema was found on the surface of some deep ulcers with black and brown membrane, hyperemia of the surrounding intestinal mucosa, edema, thickening of the intestinal wall of the lesion, and the degeneration and necrosis of mucosal epithelial cells under the rigid.HE staining, the mucosa propria, the infiltration of the mucous granulocytes in the submucosal layer accompanied with cellulose exudation and flaky bleeding. The histopathological changes of acute inflammation, similar to the changes in the acute phase of human UC, were similar to that of the.UC model group. The hypothalamic microscope showed dilatation and congestion of the cerebral vessels, the widening of the perivascular space and the shape of some nerve cells; 2) the results of the hypothalamic metabolic group showed that the significant peak metabolites of the abnormal bile fluid syndrome group were valine, leucine and brighten. Aminoacid, ethanol, gamma aminobutyric acid, N- acetyl aspartic acid, choline and inositol, significant peak metabolites with a significant peak value of the group of glutamic acid and taurine.UC: ethanol, gamma aminobutyric acid, N- acetyl aspartic acid, lactic acid, phenylalanine. By RNA-Seq analysis of the samples, the abnormal bile liquid rats were found to be in the lower level. A large number of pathways in the thalamus are associated with neural signaling pathways, such as "Neuroactive ligand-receptor interaction", "Cocaine addiction", "Amphetamine addiction", "Serotonergic synapse", "dopaminergic synapse" and so on in the hypothalamus. "", "Calcium signaling pathway", "Tyrosine metabolism", "Cytokine-cytokine receptor interaction", "PI3K-Akt signaling pathway" and other pathways. Toimmune thyroid disease ", the differentially expressed genes were Cga, Tshb. combined with GO- protein network map and gene pathway network map, and four common up-regulated genes were found, Pmch, Cga, Tshb and Hcrt; 3.) the colon metabolic group analysis showed that the abnormal bile fluid group had significant peak metabolites as: 3- hydroxy butyric acid, lactic acid, succinic, ATP/ADP.. The difference metabolite of GSH, glycerophosphoric acid choline and inositol.UC showed that the significant peak metabolites of the colon of UC group were as follows: butyric acid, propionic acid, ethanol, 3- hydroxybutyric acid, acetic acid, succinic acid, methylamine, two methylamine, malonic acid, glucose and glycogen,.UC group The significant low peak metabolites were alanine, glutamine, glutathione, glycerol phosphoric acid, inositol, creatine phosphate, taurine, creatinine. The colonic tissue obtained differential expression genes through GO and KEGG analysis, and found inflammation in the colon of abnormal bile duct group, the expression of immune related pathway gene expression was down, and UC group inflammation, immune related The phenomenon of up regulation of the pathway gene expression. The abnormal bile fluid syndrome and the UC group were set together, and the WGCNA was used to analyze it. The low expression in the normal group and the abnormal bile liquid syndrome group was selected. The high expression of module in the UC group was analyzed by KEGG pathway, and most of the functional pathways were related to the immune system, such as "Cytokine-cytokine receptor interaction", "TNF sig". Naling pathway "," PI3K-Akt signaling pathway "," NF-kappa B signaling pathway "and other signaling pathways, and also found 2 thyroid hormone related pathways, screening the main signaling pathways of thyroid hormones, through the INTERGENE interaction network diagram, found Tnf is the core molecule. Through the Thrsp gene to make a common table. The down regulation of the key enzymes PCK1, FBP and GK up-regulated, PKLR and GPD1 were downregulated in glycolysis, and the expression of fatty acid beta Co A dehydrogenase (Acadsb) gene expression was up-regulated, glutathione S transferase and glutamate cysteine ligase were all regulated; 4) T3, T4, FT3, level in abnormal bile fluid syndrome The group and the UC group were all lower, although the abnormal bile liquid syndrome group was lower, but still in the normal range, the UC group was obviously lower than the normal level. The blood glucose, fructose amine, triglyceride, apolipoprotein B were significantly increased in the UC group, and the high density lipoprotein decreased significantly (P0.05). The abnormal bile fluid syndrome group finally identified 13 different metabolic components in the rat serum, and the significant increase of metabolites in the abnormal bile duct group was isoleucine, alanine, valine, proline, pyruvic acid, carnitine, beta hydroxybutyric acid, lactic acid, and creatine, and the significantly reduced metabolites were VLDL, citrullinine and cheese. After comparison with the normal group, 13 different metabolites were found in the UC group. The significant metabolites in the UC group were LDL, lactic acid, and creatine, and the significantly reduced metabolites in the group UC were leucine, valine, citrulline, tyrosine, alanine, carnitine, citric acid, beta glucose, alpha glucose, methyl histidine. Conclusion: 1) feed through dry heat. Material, dry and hot environment feeding, tailing, noise, and chronic foot electrical stimulation multi factor compound method, rats appeared to appear emotional excitement, happy fighting, tongue fur yellow, urine yellow, dry and hard stool, weight loss, drinking water, diet increased. Successfully simulated the syndrome model which conforms to the clinical manifestations of human abnormal bile fluid syndrome. TNBS ethanol liquid enema induced ulcerative inflammation in rats. In the model, the colon tissue appeared similar to the acute phase of human UC; 2) abnormal secretion of neurotransmitter in the hypothalamus, destruction of membrane stability and abnormal energy metabolism were the common manifestations of abnormal bile liquid syndrome group and UC group, and the expression of Cga and Tshb expression of.TSH was up. It can be the response of the abnormal bile liquid syndrome group and the hypothalamus to the stimulation of the UC group. It plays a very important role in the abnormal bile fluid syndrome and the formation of UC, which plays an important role in the UC process of abnormal bile fluid syndrome. 3) the imbalance of energy metabolism, the imbalance of oxidation antioxidant system and the stability of cell membrane in the colon tissue. Destruction is the main cause of ulcerative colitis, and it is also the main cause of UC in abnormal bile duct syndrome..TNF- a, on the one hand, causes oxidative damage to colon cells by inhibiting GSH activity. On the other hand, it is a proinflammatory cytokine, activated by activation and amplification, and activated by DAG-PKC-NF- kappa B signal transduction pathway. Inflammatory response plays an important role in the formation of UC; 4) the reduction of serum thyroid hormone levels is a common material basis for abnormal bile fluid syndrome and UC groups. During the formation of abnormal bile fluid and UC, the interaction of the neuroendocrine system and the immune system, on the one hand, the immune system (TNF) acts on the nerve internal components through the cytokine (TNF). The system makes the hormone release abnormality in the nervous endocrine system (lower thyroid hormone content), on the other hand, the thyroid hormone acts on the whole body, making the whole metabolism change, such as inhibiting the metabolism of sugar, promoting the decomposition of lipids, making the energy imbalance, and causing the whole body immune disorder to cause the colon to be damaged. It is the main reason that abnormal biliary syndrome is in sub-health state and one of the reasons that abnormal biliary syndrome induces UC.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R29

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