【摘要】：背景及目的:銀屑病是一種多基因遺傳背景下發(fā)生的以角質(zhì)形成細胞和T細胞參與為主的慢性炎癥免疫性疾病,皮損以鱗屑性紅斑為主要表現(xiàn)。臨床研究發(fā)現(xiàn)銀屑病患者易出現(xiàn)胰島素抵抗,發(fā)生代謝紊亂,而一些代謝物質(zhì)的水平異�？纱龠M機體炎癥反應(yīng)。近年來,代謝組學研究表明,銀屑病患者體內(nèi)半乳糖水平顯著升高。半乳糖是糖代謝過程中產(chǎn)生的一種低分子糖,人類飲食牛奶及卡拉膠等物質(zhì)后通過半乳糖苷酶分解產(chǎn)生半乳糖,吸收進入人體的半乳糖可以在相關(guān)酶的作用下轉(zhuǎn)化為l-磷酸葡萄糖進入葡萄糖代謝途徑。當機體存在胰島素抵抗、糖代謝紊亂、糖代謝酶異�；蛘唢嬍尺^多含半乳糖的食物時,可能引起半乳糖在體內(nèi)堆積。體內(nèi)高濃度的半乳糖會誘導(dǎo)IL-6、IL-1β、TNF-α等炎癥因子的分泌,激發(fā)炎癥反應(yīng)。IL-6、IL-1β、TNF-α等炎癥因子在銀屑病患者炎癥反應(yīng)中起到重要作用,均參與銀屑病皮損角質(zhì)形成細胞增殖的過程。銀屑病患者體內(nèi)半乳糖升高的原因及半乳糖的代謝異常是否通過激活炎癥反應(yīng)參與到銀屑病的發(fā)生發(fā)展,目前缺乏相關(guān)的研究。本研究旨在:1.分析銀屑病患者體內(nèi)半乳糖水平是否較正常人升高,分析銀屑病患者體內(nèi)半乳糖水平與炎癥因子、代謝指標等的相關(guān)性;2.并通過體外實驗探討半乳糖對角質(zhì)形成細胞增殖、角質(zhì)形成細胞炎癥因子表達及PI3K/Akt通路的影響;3.檢測銀屑病患者PBMC中α-半乳糖苷酶表達水平。方法:(1)尋常型銀屑病患者半乳糖水平及其與代謝指標、炎癥因子的相關(guān)性研究:收集2015年1月至2017年2月在廣東省皮膚病醫(yī)院住院的57例尋常型銀屑病患者與同期體檢的年齡、性別匹配的28例正常對照者的各項實驗室指標及一般資料。(1)采用比色法檢測血清半乳糖水平,分析兩組半乳糖水平的差異,分析半乳糖與pasi評分、血糖、血脂、尿酸的相關(guān)性。(2)隨機選取其中38例尋常型銀屑病患者及16例正常人,用化學發(fā)光法檢測血清中空腹胰島素水平,根據(jù)穩(wěn)態(tài)模型法用空腹血糖和空腹胰島素水平計算胰島素抵抗指數(shù),比較兩組胰島素抵抗指數(shù)的差異,分析尋常型銀屑病患者血清半乳糖水平與胰島素抵抗指數(shù)的相關(guān)性。(3)隨機選取其中25例尋常型銀屑病患者及25例正常人,用elisa法檢測血清中il-6、il-1β、tnf-α、il-17、il-22、il-23的水平,分析尋常型銀屑病患者血清半乳糖水平與炎癥因子水平的相關(guān)性。(2)半乳糖對hacat細胞增殖、炎癥因子表達、pi3k/akt通路的影響研究:(1)細胞增殖實驗:用0μmol/l,200μmol/l,500μmol/l,1000μmol/l,2000μmol/l,5000μmol/l濃度的半乳糖干預(yù)hacat細胞24h,采用cck-8法觀察不同濃度的半乳糖對hacat細胞增殖的影響。(2)炎癥因子表達實驗:用0μmol/l,200μmol/l,2000μmol/l濃度的半乳糖干預(yù)hacat細胞24h,采用rt-pcr檢測炎癥因子il-1β、il-6、tnf-α的mrna表達情況。(3)pi3k/akt通路的影響實驗:用0μmol/l,200μmol/l,2000μmol/l濃度的半乳糖干預(yù)hacat細胞24h后,再用胰島素干預(yù)hacat細胞10分鐘,采用蛋白印跡法(westernblot,wb)檢測信號通路蛋白p-akt及akt的表達水平。(3)尋常型銀屑病患者pbmc中α-半乳糖苷酶mrna表達水平檢測:采用rt-pcr法檢測尋常型銀屑病患者及健康人pbmc中α-半乳糖苷酶的mrna,比較尋常型銀屑病患者與健康人pbmc中α-半乳糖苷酶的mrna表達水平有無差異。結(jié)果:(1)尋常型銀屑病患者半乳糖水平及其與代謝指標、炎癥因子的相關(guān)性研究結(jié)果:尋常型銀屑病患者的血清半乳糖水平較正常對照組顯著升高(p0.001)。尋常型銀屑病患者體內(nèi)半乳糖與甘油三酯及胰島素抵抗指數(shù)呈正相關(guān),與載脂蛋白a呈負相關(guān)(p0.05),與pasi評分、血葡萄糖、尿酸及血脂中的其它項目無明顯相關(guān)性。尋常型銀屑病患者血清中il-6、il-1β、tnf-α、il-22、il-23的水平較正常人升高(p0.05),il-17的水平無明顯差異(p0.05),尋常型銀屑病患者血清半乳糖水平與il-22水平呈正相關(guān)(p0.05),與其它炎癥因子的水平無明顯相關(guān)性。(2)細胞實驗結(jié)果:(1)細胞增殖實驗結(jié)果:0μmol/l,200μmol/l,500μmol/l,1000μmol/l,2000μmol/l,5000μmol/l濃度半乳糖處理hacat細胞24h,其中500μmol/l以上濃度組的細胞增殖率顯著高于陰性對照組(p0.05)。(2)半乳糖對hacat細胞il-6、il-1β、tnf-α的mrna表達影響的實驗結(jié)果:分別以200μmol/l、2000μmol/l濃度的半乳糖處理hacat細胞24h后,其中200μmol/l濃度的半乳糖處理組il-6的mrna表達量顯著高于陰性對照組(p0.05)。200μmol/l、2000μmol/l濃度的半乳糖處理hacat細胞24h后il-1β的mrna表達量均顯著高于對照組(p0.05)。200μmol/l、2000μmol/l濃度的半乳糖處理hacat細胞24h后tnf-α的mrna表達量與對照組無明顯差異(p0.05)。(3)半乳糖對pi3k/akt通路影響實驗的結(jié)果:以200μmol/l,2000μmol/l半乳糖干預(yù)細胞后p-akt/akt比值較對照組下降(前者p0.05,后者p0.01),且2000μmol/l濃度的半乳糖干預(yù)組較200μmol/l濃度的半乳糖干預(yù)組降低p-akt/akt比值的作用更明顯(p0.05)。(3)尋常型銀屑病患者體內(nèi)α-半乳糖苷酶mrna表達水平檢測結(jié)果:尋常型銀屑病患者血液pbmc中α-半乳糖苷酶的mrna表達水平較對照組升高(p0.05)。結(jié)論:(1)尋常型銀屑病患者血清半乳糖水平較健康對照者顯著升高,且半乳糖水平與甘油三酯、胰島素抵抗指數(shù)及炎癥因子il-22水平呈正相關(guān),與載脂蛋白a呈負相關(guān)。說明半乳糖與銀屑病患者部分代謝物質(zhì)代謝紊亂及炎癥反應(yīng)存在一定的關(guān)系。(2)一定濃度范圍內(nèi)的半乳糖能夠促進人角質(zhì)形成細胞增殖,能夠促進人角質(zhì)形成細胞細胞表達IL-6和IL-1β并且抑制胰島素信號通路中的PI3K/Akt通路。尋常型銀屑病患者體內(nèi)高濃度的半乳糖水平可能通過促進相關(guān)炎癥因子的釋放從而促進角質(zhì)增生、胰島素抵抗的發(fā)生及銀屑病患者的炎癥反應(yīng)。(3)尋常型銀屑病患者體內(nèi)α-半乳糖苷酶的mRNA表達水平較健康人明顯升高,可能是尋常型銀屑病患者體內(nèi)半乳糖水平升高的原因之一;尋常型銀屑病患者體內(nèi)半乳糖水平與胰島素抵抗指數(shù)呈正相關(guān),但具體因果關(guān)系不明確,不能排除胰島素抵抗引起的糖代謝異常導(dǎo)致半乳糖在體內(nèi)堆積的可能。
[Abstract]:Background and purpose: psoriasis is a chronic inflammatory disease characterized by the involvement of keratinocytes and T cells in the genetic background of multiple genes. The skin lesions are mainly characterized by scaly erythematous erythema. The clinical study shows that patients with psoriasis are prone to insulin resistance, metabolic disorders, and some metabolic substances in abnormal levels. In recent years, metabonomics studies have shown that the level of galactose in the patients with psoriasis is significantly increased. Galactose is a low molecular sugar produced in the process of sugar metabolism. Human milk and carrageenan can be decomposed by galactosidase to produce galactose, and the absorption of galactose into human body can be related to the relationship. The enzyme is converted into l- phosphate glucose into the glucose metabolism pathway. When the body has insulin resistance, glucose metabolism disorder, abnormal glucose metabolism enzyme, or diet too much lactose containing food, it may cause the accumulation of galactose in the body. High concentration of galactose in the body can induce the secretion of inflammatory factors such as IL-6, IL-1 beta, TNF- alpha and so on. Inflammatory reaction.IL-6, IL-1 beta, TNF- alpha and other inflammatory factors play an important role in the inflammatory response of psoriatic patients. They all participate in the proliferation of keratinocytes in psoriatic lesions. The cause of the increase of galactose in psoriatic patients and whether the metabolic abnormalities of semi lactose are involved in the development of psoriasis by activating the inflammatory reaction. The purpose of this study was to investigate whether the level of galactose in the patients with psoriasis was higher than that of normal people, and the correlation between the levels of galactose in the patients with psoriasis and inflammatory factors and metabolic indexes was analyzed. 2. and the proliferation of keratinocytes, the expression of inflammatory factors in keratinocytes and P were investigated in vitro. The effect of I3K/Akt pathway; 3. the level of alpha galactosidase expression in PBMC in patients with psoriasis. Methods: (1) the correlation between galactose level and its metabolic index and inflammatory factors in patients with psoriasis vulgaris: 57 cases of psoriasis vulgaris in Guangdong Department of Dermatology from January 2015 to February 2017 and the same period of physical examination were collected. Age, the laboratory indexes and general data of 28 normal controls. (1) the level of serum galactose was detected by colorimetry, the difference in the level of two groups of galactose was analyzed, the correlation between galactose and PASI score, blood sugar, blood lipid and uric acid were analyzed. (2) 38 cases of psoriasis vulgaris and 16 normal people were randomly selected. The level of fasting insulin in serum was detected by chemiluminescence. The insulin resistance index was calculated with fasting blood glucose and fasting insulin level by the steady state model method. The difference of insulin resistance index between the two groups was compared. The correlation between serum galactose level and insulin resistance index in patients with psoriasis vulgaris was analyzed. (3) 25 of them were randomly selected. Cases of psoriasis vulgaris and 25 normal people, the serum levels of IL-6, IL-1 beta, tnf- a, IL-17, IL-22, IL-23 were detected by ELISA. The correlation between serum galactose level and inflammatory factors in patients with psoriasis vulgaris was analyzed. (2) the effect of galactose on proliferation of HaCaT cells, expression of inflammatory factors, and pi3k/akt pathway: (1) cell proliferation Experiment: the effect of galactose on HaCaT cell 24h was interfered with 0 mu mol/l, 200 mol/l, 500 mu mol/l, 1000 mol/l, 2000 mu mol/l, and 5000 micron mol/l concentration. (2) the effect of galactose at different concentrations on the proliferation of HaCaT cells. (2) the expression of inflammatory factors: 0 micron mol/l, 200 micron mol/l, 2000 mu galactose intervened with galactose. PCR was used to detect the mRNA expression of inflammatory factors IL-1 beta, IL-6, and tnf- alpha. (3) the effect of pi3k/akt pathway: the intervention of HaCaT cells with 0 u mol/l, 200 mu mol/l, 2000 micron concentration of galactose was used to intervene HaCaT cells for 10 minutes, and the expression level of signaling pathway protein was detected by Western blotting. (3 The expression level of alpha galactosidase mRNA in PBMC of patients with psoriasis vulgaris: RT-PCR method was used to detect the mRNA of alpha galactosidase in psoriasis vulgaris and healthy people PBMC. There was no difference in the mRNA expression level of alpha galactosidase in patients with psoriasis vulgaris and healthy people PBMC. Results: (1) half of patients with psoriasis vulgaris Lactose level and its correlation with metabolic indices and inflammatory factors: Serum galactose levels in patients with psoriasis vulgaris were significantly higher than those in normal controls (p0.001). In patients with psoriasis vulgaris, galactose and triglyceride and insulin resistance index Cheng Zhengxiang, and apolipoprotein A were negatively correlated (P0.05), and PASI scores, There was no significant correlation between blood glucose, uric acid and other items in blood lipids. The levels of IL-6, IL-1 beta, tnf- a, IL-22, IL-23 in patients with psoriasis vulgaris were higher than those of normal people (P0.05), and there was no significant difference in the level of IL-17 (P0.05). The level of serum galactose in psoriasis vulgaris patients was positively correlated with the level of IL-22 (P0.05), and other inflammatory causes. (2) the results of cell experiment: (1) the results of cell proliferation experiment: 0 mu mol/l, 200 mu mol/l, 500 mu mol/l, 1000 mu mol/l, 2000 mu mol/l, and 5000 micron galactose to treat HaCaT cell 24h, and the proliferation rate of 500 u mol/l above the concentration group was significantly higher than that of the negative control group (P0.05). (2) galactose to HaCaT cell IL-6. The experimental results of the effect of mRNA expression of L-1 beta, tnf- alpha on the treatment of HaCaT cell 24h with 200 u mol/l and 2000 mol/l concentration of galactose respectively, the mRNA expression of IL-6 in the galactose treatment group with 200 mu mol/l concentration was significantly higher than that of the negative control group (P0.05).200 muon. The amount of HaCaT cells was significantly higher than that of the control group (P0.05).200 mol/l, and the mRNA expression of tnf- alpha in HaCaT cells treated with galactose at 2000 mol/l concentration was not significantly different from that of the control group (P0.05). (3) the effect of galactose on the pi3k/akt pathway: 200 Mu mol/l, 2000 micron half lactose intervened cells after the reduction of the ratio compared with the control group. 0.05, the latter P0.01), and the effect of reducing the p-akt/akt ratio in the galactose intervention group with the concentration of 2000 mu mol/l was more obvious (P0.05). (3) the level of alpha galactosidase mRNA expression in the patients with psoriasis vulgaris: the mRNA expression water of the alpha galactosidase in the blood PBMC of patients with vulgaris disease. Compared with the control group (P0.05). Conclusion: (1) the level of serum galactose in patients with psoriasis vulgaris was significantly higher than that of healthy controls, and the level of galactose was positively correlated with the level of triglyceride, insulin resistance index and inflammatory factor IL-22, and negative correlation with apolipoprotein A. There is a certain relationship between the disorder and the inflammatory response. (2) the galactose within a certain concentration can promote the proliferation of human keratinocytes, promote the expression of IL-6 and IL-1 beta in human keratinocyte cells and inhibit the PI3K/Akt pathway in the insulin signaling pathway. Promoting the release of related inflammatory factors to promote keratinosis, insulin resistance and inflammatory response in patients with psoriasis. (3) the level of mRNA expression of alpha galactosidase in psoriasis vulgaris is significantly higher than that in healthy people, and may be one of the reasons for the increase in the level of intra - body galactose in patients with psoriasis vulgaris; The level of galactose in the patients with chip disease is positively correlated with the insulin resistance index, but the specific causality is not clear. It can not exclude the possibility of the accumulation of galactose in the body due to the abnormal glucose metabolism caused by insulin resistance.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R758.63

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