【摘要】：目的:本研究論文主要選擇維醫(yī)異常膽液質(zhì)證模型大鼠與TNBS-乙醇灌腸液誘導(dǎo)的潰瘍性結(jié)腸炎模型大鼠為研究對(duì)象,通過(guò)分析大鼠一般體征、下丘腦及結(jié)腸組織形態(tài)學(xué)結(jié)構(gòu)變化;通過(guò)代謝組學(xué)分析下丘腦、結(jié)腸代謝組份差異;通過(guò)RNA-Seq測(cè)序技術(shù)研究下丘腦、結(jié)腸轉(zhuǎn)錄譜差異;通過(guò)研究甲狀腺激素異常對(duì)異常膽液質(zhì)證與UC大鼠代謝紊亂產(chǎn)生的作用,綜合分析異常膽液質(zhì)證形成及其誘發(fā)UC的物質(zhì)基礎(chǔ)。方法:1)采用干熱飼養(yǎng)環(huán)境、干熱屬性飼料(黑胡椒、干姜、西紅花)、夾尾、噪音等多因素復(fù)合刺激,建立異常膽液質(zhì)證載體動(dòng)物模型。通過(guò)TNBS乙醇液灌腸復(fù)制大鼠潰瘍性結(jié)腸炎模型,觀察大鼠生物表型改變及組織形態(tài)學(xué)改變;2)利用核磁共振技術(shù)檢測(cè)多因素復(fù)合誘導(dǎo)的異常膽液質(zhì)證、UC大鼠模型中下丘腦和結(jié)腸代謝產(chǎn)物的變化;3)應(yīng)用RNA-seq全面分析異常膽液質(zhì)證和UC大鼠下丘腦和結(jié)腸組織的轉(zhuǎn)錄本,篩選出差異表達(dá)基因。第二,應(yīng)用GO方法進(jìn)行差異基因的功能聚類,應(yīng)用KEGG信號(hào)通路富集差異表達(dá)基因相關(guān)的信號(hào)通路。第三,構(gòu)建蛋白質(zhì)相互作用網(wǎng)絡(luò),分析差異表達(dá)基因之間的相互作用,篩選出重要的差異表達(dá)基因。第四,應(yīng)用實(shí)時(shí)熒光定量PCR方法驗(yàn)證差異基因的表達(dá)水平;4)檢測(cè)血清中甲狀腺功能7項(xiàng)指標(biāo)及血清糖脂代謝相關(guān)生化指標(biāo),通過(guò)相關(guān)性分析,分析甲狀腺激素水平與異常膽液質(zhì)證及潰瘍性結(jié)腸炎形成中對(duì)代謝的影響。同時(shí)通過(guò)血清代謝組學(xué)的結(jié)果繪制代謝的整體網(wǎng)絡(luò)調(diào)控圖。結(jié)果:1)通過(guò)多因素復(fù)合造模方法成功模擬出了符合人體異常膽液質(zhì)證臨床表現(xiàn)的證候模型以及成功復(fù)制了TNBS-UC模型。肉眼觀察異常膽液質(zhì)證結(jié)腸組織出現(xiàn)輕微充血,HE染色后鏡下觀察,發(fā)現(xiàn)異常膽液質(zhì)證組出現(xiàn)少量炎細(xì)胞浸潤(rùn)。異常膽液質(zhì)證組下丘腦鏡下出現(xiàn)腦血管擴(kuò)張充血,血管周圍間隙增寬,部分神經(jīng)細(xì)胞形態(tài)變圓。UC組結(jié)腸肉眼可見腸壁壞死充血水腫現(xiàn)象,部分深大潰瘍表面有黑褐色膜狀物附著,周邊腸黏膜充血、水腫明顯,病變部位腸壁明顯增厚,管壁僵硬。HE染色后鏡下可見黏膜上皮細(xì)胞變性壞死,黏膜固有層,黏膜下層大量中性粒細(xì)胞浸潤(rùn)伴纖維素滲出及片狀出血,符合急性炎癥的組織病理學(xué)改變,與人類UC急性期改變相似。UC模型組下丘腦鏡下出現(xiàn)腦血管擴(kuò)張充血,血管周圍間隙增寬,部分神經(jīng)細(xì)胞形態(tài)變圓;2)下丘腦代謝組學(xué)研究結(jié)果顯示異常膽液質(zhì)證組顯著性高峰度代謝物為纈氨酸、亮氨酸、異亮氨酸、乙醇、γ-氨基丁酸、N-乙酰天冬氨酸、膽堿和肌醇,顯著性低峰度代謝物為谷氨酸和�；撬帷C組具有顯著性高峰度代謝物為:乙醇,γ-氨基丁酸,N-乙酰天冬氨酸,乳酸,苯丙氨酸。通過(guò)對(duì)樣本間進(jìn)行RNA-Seq分析,發(fā)現(xiàn)異常膽液質(zhì)證大鼠在下丘腦中大量的通路與神經(jīng)信號(hào)通路相關(guān),如“Neuroactive ligand-receptor interaction”,“Cocaine addiction”,“Amphetamine addiction”,“Serotonergic synapse”,“dopaminergic synapse”等。UC組在丘腦組織中上調(diào)表達(dá)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。異常膽液質(zhì)證組和UC組差異基因共表達(dá)分析,發(fā)現(xiàn)2條和甲狀腺激素應(yīng)答相關(guān)通路,分別是“Thyroid hormone synthesis”、“Autoimmune thyroid disease”,差異表達(dá)基因?yàn)镃ga,Tshb。結(jié)合GO-蛋白網(wǎng)絡(luò)圖和基因通路網(wǎng)絡(luò)圖,找到四個(gè)共同上調(diào)基因,分別是Pmch、Cga、Tshb和Hcrt;3)結(jié)腸代謝組學(xué)分析顯示異常膽液質(zhì)證組具有顯著性高峰度代謝物為:3-羥基丁酸,乳酸,琥珀酸,ATP/ADP。異常膽液質(zhì)證組具有顯著性低峰度代謝物為:GSH,甘油磷酸膽堿,肌醇。UC組的差異代謝物結(jié)果顯示,和正常組對(duì)比,UC組結(jié)腸具有顯著性高峰度代謝物為:丁酸,丙酸,乙醇,3-羥基丁酸,乙酸,琥珀酸,甲胺,二甲胺,丙二酸,葡萄糖,糖原。UC組具有顯著性低峰度代謝物為:丙氨酸,谷氨酰胺,谷胱甘肽,甘油磷酸膽堿,肌醇,磷酸肌酸,�；撬�,肌酐。結(jié)腸組織獲得差異表達(dá)基因通過(guò)GO和KEGG分析,發(fā)現(xiàn)在異常膽液質(zhì)證組結(jié)腸出現(xiàn)了炎癥、免疫相關(guān)通路基因表達(dá)下調(diào),而UC組炎癥、免疫相關(guān)通路基因表達(dá)上調(diào)的現(xiàn)象。將異常膽液質(zhì)證與UC組取并集,運(yùn)用WGCNA進(jìn)行分析,選擇在正常組和異常膽液質(zhì)證組低表達(dá),UC組高表達(dá)的module進(jìn)行KEGG通路分析,得到的大部分功能通路與免疫相關(guān),如“Cytokine-cytokine receptor interaction”,“TNF signaling pathway”,“PI3K-Akt signaling pathway”,“NF-kappa B signaling pathway”等信號(hào)通路,同時(shí)也發(fā)現(xiàn)2條甲狀腺激素相關(guān)的通路,篩選甲狀腺激素主要信號(hào)通路,通過(guò)基因間的相互作用網(wǎng)路關(guān)系圖,發(fā)現(xiàn)Tnf是其中的核心分子。通過(guò)Thrsp基因做共表達(dá)分析,發(fā)現(xiàn)糖異生關(guān)鍵酶PCK1、FBP的下調(diào)和GK上調(diào)。同時(shí)糖酵解途徑中PKLR和GPD1出現(xiàn)下調(diào),脂肪酸β-氧化中脂酰Co A脫氫酶(Acadsb)基因表達(dá)卻上調(diào),谷胱甘肽S轉(zhuǎn)移酶和谷氨酸-半胱氨酸連接酶均下調(diào);4)血清中T3、T4、FT3、FT4水平在異常膽液質(zhì)證組和UC組均降低,異常膽液質(zhì)證組雖然降低,但仍然處在正常范圍內(nèi),UC組明顯低于正常水平。血糖、果糖胺、甘油三酯、載脂蛋白B在UC組中顯著升高,高密度脂蛋白顯著降低,(P0.05)。甲狀腺激素與葡萄糖、甘油三酯、總膽固醇存在負(fù)相關(guān),與高密度膽固醇正相關(guān)。異常膽液質(zhì)證組最終確定大鼠血清中有13種的差異性代謝成分,其中異常膽液質(zhì)證組中顯著升高的代謝物為:異亮氨酸、丙氨酸、纈氨酸、脯氨酸、丙酮酸、肉堿、β-羥丁酸、乳酸、肌酸,顯著降低的代謝物為:VLDL、瓜氨酸、酪氨酸。和正常組比較后,UC組中共找到13種差異代謝物,在UC組中顯著升高的代謝物為:LDL、乳酸、肌酸,UC組中顯著降低的代謝物為:亮氨酸、纈氨酸、瓜氨酸、酪氨酸、丙氨酸、肉堿、檸檬酸、β-葡萄糖、α-葡萄糖、甲基組氨酸。結(jié)論:1)通過(guò)干熱屬性飼料,干熱環(huán)境喂養(yǎng),夾尾、噪音、慢性足底電刺激多因素復(fù)合方法造模,大鼠出現(xiàn)了出現(xiàn)情緒易激動(dòng)、喜打斗、舌苔發(fā)黃、尿色黃、糞便干硬、體重減輕、飲水、飲食量增加的現(xiàn)象。成功模擬出了符合人體異常膽液質(zhì)證臨床表現(xiàn)的證候模型。成功復(fù)制TNBS乙醇液灌腸誘導(dǎo)大鼠潰瘍性炎癥的發(fā)生。模型中結(jié)腸組織出現(xiàn)了與人類UC急性期相似的病理改變;2)下丘腦神經(jīng)遞質(zhì)分泌異常,膜穩(wěn)定性破壞及能量代謝異常是異常膽液質(zhì)證組與UC組中的共同表現(xiàn)。TSH編碼基因Cga和Tshb表達(dá)上調(diào),可能是異常膽液質(zhì)證組和UC組下丘腦對(duì)刺激的應(yīng)答反應(yīng),在異常膽液質(zhì)證和UC形成過(guò)成中都起到非常關(guān)鍵的作用,推測(cè)在異常膽液質(zhì)證誘發(fā)UC過(guò)程中發(fā)揮重要作用;3)結(jié)腸組織出現(xiàn)的能量代謝失衡、氧化抗氧化體系平衡紊亂、細(xì)胞膜穩(wěn)定性破壞是導(dǎo)致潰瘍性結(jié)腸炎發(fā)生的主要因素,同時(shí)也是異常膽液質(zhì)證誘發(fā)UC的主要原因。TNF-α一方面通過(guò)抑制GSH活性使結(jié)腸細(xì)胞產(chǎn)生氧化損傷,另一方面做為促炎細(xì)胞因子,通過(guò)激活和放大的雙重作用,以DAG-PKC-NF-κB信號(hào)傳導(dǎo)途徑激活而產(chǎn)生炎癥反應(yīng),在UC形成中起重要作用;4)血清甲狀腺激素含量降低是異常膽液質(zhì)證組與UC組發(fā)生的共同物質(zhì)基礎(chǔ)。異常膽液質(zhì)證與UC形成過(guò)程中,神經(jīng)內(nèi)分泌系統(tǒng)和免疫系統(tǒng)相互作用,一方面免疫系統(tǒng)通過(guò)細(xì)胞因子(TNF)作用于神經(jīng)-內(nèi)分泌系統(tǒng),使神經(jīng)-內(nèi)分泌系統(tǒng)激素釋放異常(甲狀腺激素含量降低),另一方面甲狀腺激素又作用于全身,使整體代謝發(fā)生改變?nèi)缫种铺谴x,促進(jìn)脂類分解,使能量失衡,并產(chǎn)生全身免疫紊亂使結(jié)腸受到損傷。異常膽液質(zhì)證組血清甲狀腺激素含量偏低但仍處于正常范圍,是異常膽液質(zhì)證處于亞健康狀態(tài)的主要原因,也是異常膽液質(zhì)證誘發(fā)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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R29

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