半乳糖对寻常型银屑病炎症反应及角质形成细胞增殖的影响
发布时间：2018-07-26 17:49
【摘要】：背景及目的:银屑病是一种多基因遗传背景下发生的以角质形成细胞和T细胞参与为主的慢性炎症免疫性疾病,皮损以鳞屑性红斑为主要表现。临床研究发现银屑病患者易出现胰岛素抵抗,发生代谢紊乱,而一些代谢物质的水平异常可促进机体炎症反应。近年来,代谢组学研究表明,银屑病患者体内半乳糖水平显著升高。半乳糖是糖代谢过程中产生的一种低分子糖,人类饮食牛奶及卡拉胶等物质后通过半乳糖苷酶分解产生半乳糖,吸收进入人体的半乳糖可以在相关酶的作用下转化为l-磷酸葡萄糖进入葡萄糖代谢途径。当机体存在胰岛素抵抗、糖代谢紊乱、糖代谢酶异常或者饮食过多含半乳糖的食物时,可能引起半乳糖在体内堆积。体内高浓度的半乳糖会诱导IL-6、IL-1β、TNF-α等炎症因子的分泌,激发炎症反应。IL-6、IL-1β、TNF-α等炎症因子在银屑病患者炎症反应中起到重要作用,均参与银屑病皮损角质形成细胞增殖的过程。银屑病患者体内半乳糖升高的原因及半乳糖的代谢异常是否通过激活炎症反应参与到银屑病的发生发展,目前缺乏相关的研究。本研究旨在:1.分析银屑病患者体内半乳糖水平是否较正常人升高,分析银屑病患者体内半乳糖水平与炎症因子、代谢指标等的相关性;2.并通过体外实验探讨半乳糖对角质形成细胞增殖、角质形成细胞炎症因子表达及PI3K/Akt通路的影响;3.检测银屑病患者PBMC中α-半乳糖苷酶表达水平。方法:(1)寻常型银屑病患者半乳糖水平及其与代谢指标、炎症因子的相关性研究:收集2015年1月至2017年2月在广东省皮肤病医院住院的57例寻常型银屑病患者与同期体检的年龄、性别匹配的28例正常对照者的各项实验室指标及一般资料。(1)采用比色法检测血清半乳糖水平,分析两组半乳糖水平的差异,分析半乳糖与pasi评分、血糖、血脂、尿酸的相关性。(2)随机选取其中38例寻常型银屑病患者及16例正常人,用化学发光法检测血清中空腹胰岛素水平,根据稳态模型法用空腹血糖和空腹胰岛素水平计算胰岛素抵抗指数,比较两组胰岛素抵抗指数的差异,分析寻常型银屑病患者血清半乳糖水平与胰岛素抵抗指数的相关性。(3)随机选取其中25例寻常型银屑病患者及25例正常人,用elisa法检测血清中il-6、il-1β、tnf-α、il-17、il-22、il-23的水平,分析寻常型银屑病患者血清半乳糖水平与炎症因子水平的相关性。(2)半乳糖对hacat细胞增殖、炎症因子表达、pi3k/akt通路的影响研究:(1)细胞增殖实验:用0μmol/l,200μmol/l,500μmol/l,1000μmol/l,2000μmol/l,5000μmol/l浓度的半乳糖干预hacat细胞24h,采用cck-8法观察不同浓度的半乳糖对hacat细胞增殖的影响。(2)炎症因子表达实验:用0μmol/l,200μmol/l,2000μmol/l浓度的半乳糖干预hacat细胞24h,采用rt-pcr检测炎症因子il-1β、il-6、tnf-α的mrna表达情况。(3)pi3k/akt通路的影响实验:用0μmol/l,200μmol/l,2000μmol/l浓度的半乳糖干预hacat细胞24h后,再用胰岛素干预hacat细胞10分钟,采用蛋白印迹法(westernblot,wb)检测信号通路蛋白p-akt及akt的表达水平。(3)寻常型银屑病患者pbmc中α-半乳糖苷酶mrna表达水平检测:采用rt-pcr法检测寻常型银屑病患者及健康人pbmc中α-半乳糖苷酶的mrna,比较寻常型银屑病患者与健康人pbmc中α-半乳糖苷酶的mrna表达水平有无差异。结果:(1)寻常型银屑病患者半乳糖水平及其与代谢指标、炎症因子的相关性研究结果:寻常型银屑病患者的血清半乳糖水平较正常对照组显著升高(p0.001)。寻常型银屑病患者体内半乳糖与甘油三酯及胰岛素抵抗指数呈正相关,与载脂蛋白a呈负相关(p0.05),与pasi评分、血葡萄糖、尿酸及血脂中的其它项目无明显相关性。寻常型银屑病患者血清中il-6、il-1β、tnf-α、il-22、il-23的水平较正常人升高(p0.05),il-17的水平无明显差异(p0.05),寻常型银屑病患者血清半乳糖水平与il-22水平呈正相关(p0.05),与其它炎症因子的水平无明显相关性。(2)细胞实验结果:(1)细胞增殖实验结果:0μmol/l,200μmol/l,500μmol/l,1000μmol/l,2000μmol/l,5000μmol/l浓度半乳糖处理hacat细胞24h,其中500μmol/l以上浓度组的细胞增殖率显著高于阴性对照组(p0.05)。(2)半乳糖对hacat细胞il-6、il-1β、tnf-α的mrna表达影响的实验结果:分别以200μmol/l、2000μmol/l浓度的半乳糖处理hacat细胞24h后,其中200μmol/l浓度的半乳糖处理组il-6的mrna表达量显著高于阴性对照组(p0.05)。200μmol/l、2000μmol/l浓度的半乳糖处理hacat细胞24h后il-1β的mrna表达量均显著高于对照组(p0.05)。200μmol/l、2000μmol/l浓度的半乳糖处理hacat细胞24h后tnf-α的mrna表达量与对照组无明显差异(p0.05)。(3)半乳糖对pi3k/akt通路影响实验的结果:以200μmol/l,2000μmol/l半乳糖干预细胞后p-akt/akt比值较对照组下降(前者p0.05,后者p0.01),且2000μmol/l浓度的半乳糖干预组较200μmol/l浓度的半乳糖干预组降低p-akt/akt比值的作用更明显(p0.05)。(3)寻常型银屑病患者体内α-半乳糖苷酶mrna表达水平检测结果:寻常型银屑病患者血液pbmc中α-半乳糖苷酶的mrna表达水平较对照组升高(p0.05)。结论:(1)寻常型银屑病患者血清半乳糖水平较健康对照者显著升高,且半乳糖水平与甘油三酯、胰岛素抵抗指数及炎症因子il-22水平呈正相关,与载脂蛋白a呈负相关。说明半乳糖与银屑病患者部分代谢物质代谢紊乱及炎症反应存在一定的关系。(2)一定浓度范围内的半乳糖能够促进人角质形成细胞增殖,能够促进人角质形成细胞细胞表达IL-6和IL-1β并且抑制胰岛素信号通路中的PI3K/Akt通路。寻常型银屑病患者体内高浓度的半乳糖水平可能通过促进相关炎症因子的释放从而促进角质增生、胰岛素抵抗的发生及银屑病患者的炎症反应。(3)寻常型银屑病患者体内α-半乳糖苷酶的mRNA表达水平较健康人明显升高,可能是寻常型银屑病患者体内半乳糖水平升高的原因之一;寻常型银屑病患者体内半乳糖水平与胰岛素抵抗指数呈正相关,但具体因果关系不明确,不能排除胰岛素抵抗引起的糖代谢异常导致半乳糖在体内堆积的可能。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.63
本文编号：2146806
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R758.63
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