基于代谢组学方法探讨寻常型银屑病的生物标志物并研究药物对标志物的作用

发布时间：2018-05-07 00:29

本文选题：寻常型银屑病 + 代谢组学　；参考：《暨南大学》2017年硕士论文

【摘要】：研究背景:寻常型银屑病是一种常见易复发的慢性炎症性皮肤病,目前临床上对于该病的诊断主要是根据患者的临床表现、发病部位以及组织病理学变化等。但是组织病理学检查具有一定的创伤性,实验室常规检查指标对寻常型银屑病的特异性和敏感度较低,临床价值和意义有限,因此临床上尚缺少特异性强的寻常型银屑病诊断标准。此外,该病的病因复杂,发病机制尚未完全明确,患者常伴有多种内源性代谢紊乱。而代谢组学是通过考察生物体系受刺激或扰动后(例如某个基因变异或某一病理生理状态下)代谢产物图谱及其动态变化来研究生物体系的代谢网络的一种技术,近年来已经在疾病诊断、发病机制研究、个体化给药以及药物开发等领域得到了广泛的应用。因此应用代谢组学技术对寻常型银屑病患者内源性代谢物进行研究并结合多元统计分析进行模式识别,得到的生物标志物将有助于疾病的诊断、发病机制的深入研究以及个体化给药方案的制定。甲氨蝶呤(methotrexate,MTX)和雷公藤多苷是用于银屑病治疗的常用药物,主要通过免疫抑制来发挥治疗作用,但是其副作用明显以及个体化差异较大。寻常型银屑病是一种内源性代谢物发生紊乱的疾病,通过观察MTX和雷公藤多苷对体内生物标志物的影响,可以从代谢物角度出发来研究其用于银屑病治疗的作用,这将有助于药效学指标的建立,从而为后期药物剂量的调整以及个体化给药提供依据。研究目的:1.基于代谢组学技术探讨寻常型银屑病的生物学标志物。2.探讨MTX和雷公藤多苷对银屑病小鼠与患者相匹配标志物的影响。研究方法:1.样品的收集与处理选取在暨南大学附属第一医院皮肤科治疗且符合纳入标准的寻常型银屑病患者为病例组,以体检中心的健康志愿者为对照组。清晨空腹抽取患者和健康志愿者的血液样本,置于5 m L EDTA-K2抗凝管中,采血后立即轻轻颠倒混匀5~6次,静置2h,离心(4000 r/min,15min,4℃),取上清液于-80℃冰箱冻存待测。2.基于~1H-NMR和UPLC/Q-TOF MS两种技术的血浆代谢组学研究分别采用核磁共振(nuclear magnetic resonance ~1H spectroscopy,~1H-NMR)和超高效液相色谱串联四级杆飞行时间质谱仪(ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry,UPLC/Q-TOF MS)两种技术对寻常型银屑病患者和健康志愿者的血浆样本进行代谢组学研究,同时结合主成分分析(principal component analysis,PCA)和正交偏最小二乘法判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA)进行模式识别,对OPLS-DA模型中提取的变量重要性投影(Variable Importance in Projection,VIP)值1的变量进行独立样本t检验,p0.05为差异有统计学意义,从而筛选出潜在的生物标志物并进行相关代谢途径分析。然后对这两种分析方法的结果进行比较,找到两者潜在生物标志物中的共性以及特异性指标。3.IMQ诱导小鼠银屑病模型的建立以及模型组与患者生物标志物的匹配将24只雌性BALB/c小鼠做为实验对象,适应性喂养3d后用4%水合氯醛(0.1ml/10g)腹腔注射麻醉,剔除其背部毛发,形成约2cm×3cm大小的暴露区域,随机分为2组,每组12只,分别为模型组和空白组。模型组每日在小鼠裸露背部涂抹5%咪喹莫特(imiquimod,IMQ)乳膏62.5mg,空白组涂抹等量凡士林,两组同时用生理盐水灌胃,0.2m L/次/天,连续7天。各组小鼠于第8天取材,通过肉眼观察以及病理学检查判断小鼠是否出现银屑病样皮损。然后基于UPLC/Q-TOF MS代谢组学技术对模型组和空白组小鼠血浆样本进行代谢组学分析,寻找小鼠银屑病模型中与寻常型银屑病患者相匹配的潜在生物标志物。4.MTX和雷公藤多苷对银屑病小鼠皮损部位以及相匹配标志物的影响将24只雌性BALB/c小鼠做为实验对象,适应性喂养3d后用4%水合氯醛(0.1ml/10g)腹腔注射麻醉,剔除其背部毛发,形成约2cm×3cm大小的暴露区域,随机分为2组,每组12只,分别为MTX组和雷公藤多苷组。MTX组每日在小鼠裸露背部涂抹5%IMQ乳膏62.5mg,同时以MTX(1mg/kg/d)灌胃,连续7天。雷公藤多苷组每日在小鼠裸露背部涂抹5%IMQ乳膏62.5mg,同时以雷公藤多苷(10mg/kg/d)灌胃,连续7天。各组小鼠于第8天取材,通过肉眼初步判断小鼠银屑病样皮损是否改善,然后基于UPLC/Q-TOF MS代谢组学技术探讨MTX和雷公藤多苷对银屑病小鼠中与患者相匹配标志物的影响。研究结果:1.基于~1H-NMR和UPLC/Q-TOF MS两种技术的血浆代谢组学研究分别采用~1H-NMR和UPLC/Q-TOF MS两种技术对寻常型银屑病患者和健康志愿者血浆进行代谢组学研究,结果显示两组能够被明显区分,组间内源性代谢物的含量存在明显差异。通过~1H-NMR代谢组学技术结合多元统计分析方法找到了21种差异性代谢物,分别为极低密度脂蛋白(very low density lipoprotein,VLDL)、低密度脂蛋白(low density lipoprotein,LDL)、脂类、亮氨酸、异亮氨酸、缬氨酸、β-羟基丁酸、乳酸、丙氨酸、精氨酸、N-乙酰糖蛋白、乙酰乙酸、谷氨酸、丙酮酸、谷氨酰胺、柠檬酸、肌酸、肌酐、葡萄糖、天冬氨酸和苯丙氨酸。通过UPLC/Q-TOF MS代谢组学技术结合多元统计分析方法找到了19种差异性代谢物,分别为苏氨酸、亮氨酸、苯丙氨酸、色氨酸、棕榈酸酰胺、亚油酸酰胺、油酸酰胺、硬脂酸酰胺、顺式-11-二十烯酰胺、反式-13-二十二烯酰胺、尿酸、溶血磷脂酰胆碱(lyso-phosphatidylcholine,Lyso PC)(16:0)、Lyso PC(18∶3)、Lyso PC(18:2)、Lyso PC(18:1)、Lyso PC(18:0)、油酸、花生四烯酸和N-亚油酰基牛磺酸。从以上结果可以看出,两种代谢组学技术都具有很好的聚类效果,但是得到的差异性代谢物大部分不同。2.IMQ诱导小鼠银屑病模型的建立以及模型组与患者生物标志物的匹配造模第8天通过肉眼观察可以看出,空白组小鼠背部皮肤表面光滑,模型组小鼠背部皮肤红斑、鳞屑现象明显,皮肤增厚严重。通过病理学检查可以看出,空白组小鼠皮肤表皮层很薄,细胞形态正常,模型组小鼠皮肤出现角化过度、角化不全、棘层增厚、皮突延长以及大量的炎性细胞浸润等组织病理学改变,类似银屑病样皮损,初步显示造模成功。采用UPLC/Q-TOF MS代谢组学技术对模型组和空白组小鼠血浆样本进行代谢组学分析发现,在银屑病小鼠的潜在生物标志物中,苏氨酸、苯丙氨酸、色氨酸、棕榈酸酰胺、油酸酰胺、油酸、硬脂酰胺、反式-13-二十二烯酰胺、Lyso PC(16:0)、Lyso PC(18:3)、Lyso PC(18:2)和Lyso PC(18:0)是与寻常型银屑病患者的生物标志物相匹配的。从以上结果可以看出,银屑病小鼠与寻常型银屑病患者的生物标志物具有一定的相似性。3.MTX和雷公藤多苷对银屑病小鼠皮损部位以及相匹配标志物的影响给药第8天通过肉眼观察可以看出,MTX组和雷公藤多苷组小鼠背部皮肤的红斑、鳞屑以及增厚现象与模型组相比均有所改善,表明药物能明显改善银屑病小鼠的皮损症状。进一步采用UPLC/Q-TOF MS技术结合多元统计分析对空白组、模型组、MTX组和雷公藤多苷组的内源性代谢物进行研究发现,给药组与模型组能够明显区分,并有向空白组偏移的趋势。在银屑病小鼠与患者相匹配的生物标志物中,MTX组中的苯丙氨酸、Lyso PC(16:0)、Lyso PC(18:2)和Lyso PC(18:0)含量降低,与模型组小鼠相比有统计学差异(P0.05),而与空白组小鼠相比无统计学差异(P㧐0.05);雷公藤多苷组中的苯丙氨酸、色氨酸、Lyso PC(16:0)、Lyso PC(18:2)和Lyso PC(18:0)含量降低,与模型组小鼠相比有统计学差异(P0.05),而与空白组小鼠相比无统计学差异(P㧐0.05)。从以上结果可以看出,MTX与雷公藤多苷的治疗作用可能与影响氨基酸以及LPCs的水平有关。研究结论:~1H-NMR和UPLC/Q-TOF MS技术做为代谢组学研究中的两种常用方法,在生物标志物发掘中存在一定的互补性。本文采用~1H-NMR和UPLC/Q-TOF MS两种技术对寻常型银屑病患者以及健康志愿者的内源性代谢物进行研究都具有很好的聚类效果。通过代谢组学技术结合多元统计分析找到的生物标志物将有助于疾病的诊断、发病机制的深入研究以及个体化给药方案的制定。采用IMQ可以成功诱导小鼠产生银屑病样皮损,进一步采用UPLC/Q-TOF MS代谢组学技术探讨MTX和雷公藤多苷对银屑病小鼠中与患者相匹配标志物的影响时发现,MTX与雷公藤多苷的治疗作用可能与影响氨基酸以及LPCs的水平有关。这将有助于药效学指标的建立,从而为后期药物剂量的调整以及个体化给药提供依据。
[Abstract]:Background: psoriasis vulgaris is a common and recurrent chronic inflammatory dermatosis. The diagnosis of this disease is mainly based on the clinical manifestations, the location of the patients and the histopathological changes. However, the histopathological examination has a certain trauma, and the routine examination of the laboratory is for psoriasis vulgaris. The specificity and sensitivity are low and the clinical value and significance are limited. Therefore, there is still a lack of specific diagnostic criteria for psoriasis vulgaris. In addition, the etiology of the disease is complex, the pathogenesis is not completely clear, and the patients often have many endogenous metabolic disorders. A technique for studying metabolic networks of biological systems, such as a gene mutation or a certain pathophysiological state, and its dynamic changes, has been widely used in the fields of disease diagnosis, pathogenesis, individualized administration and drug development in recent years. Therefore, the application of metabonomics to the common type has been applied. The endogenous metabolites of patients with psoriasis are studied and combined with multivariate statistical analysis for pattern recognition. The biomarkers obtained will contribute to the diagnosis of disease, the in-depth study of the pathogenesis and the formulation of the individualized drug delivery scheme. Methotrexate (MTX) He Lei rattan glucoside is a common drug used in the treatment of psoriasis. It is necessary to play a therapeutic role by immunosuppression, but its side effects and individualized differences are significant. Psoriasis vulgaris is a disorder of endogenous metabolites. By observing the effects of MTX and Tripterygium wilfordii on biomarkers, it can be used to study the treatment of psoriasis from the angle of metabolites. This will contribute to the establishment of pharmacodynamic indicators, thus providing evidence for later drug dose adjustment and individualized administration. Objective: 1. based on metabonomics, the biological marker of psoriasis vulgaris.2. and the effect of MTX and Tripterygium wilfordii polysaccharide on the matched markers of psoriasis in mice and patients. 1. Samples were collected and treated in the Department of Dermatology, the Department of Dermatology, the First Affiliated Hospital of Jinan University, which was treated in the Department of dermatology at the First Affiliated Hospital of Jinan University. The healthy volunteers in the medical center were used as the control group. The blood samples from the patients and the healthy volunteers in the early morning were placed in the 5 m L EDTA-K2 anticoagulant tube, and immediately after the blood was collected, the blood was gently reversed. Mixing 5~6 times, placing 2h, centrifuging (4000 r/min, 15min, 4 C), taking the supernatant at -80 centigrade refrigerators and cryopreservation for.2. based on the two techniques of ~1H-NMR and UPLC/Q-TOF MS, using nuclear magnetic resonance (nuclear magnetic resonance) and ultra high performance liquid chromatography in series four grade rod time of flight mass spectrometry Two techniques (ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry, UPLC/Q-TOF MS) were used to study the plasma samples of patients with psoriasis vulgaris and healthy volunteers, combined with principal component analysis (principal component) and orthogonal partial least square method (orthogonal partial least squares discriminant analysis, OPLS-DA) is used for pattern recognition, and the variable importance projection of the OPLS-DA model (Variable Importance in Projection,) value 1 is tested by independent samples, and the difference is statistically significant, thus screening out potential biomarkers and making correlation. Analysis of metabolic pathways, and then compare the results of the two analysis methods, find the commonness of the potential biomarkers and the specificity index.3.IMQ induced mouse psoriasis model and the matching of the model group and the patient biomarker. 24 female BALB/c mice are used as the experimental objects, and the adaptive feeding of 3D is 4%. Chloral hydrate (0.1ml/10g) was intraperitoneally injected into 2 groups of 12 rats in each group, each of which was a model group and a blank group. The model group was smeared with 5% imiquimod (imiquimod, IMQ) cream 62.5mg, the blank group was applied to the same amount of Vaseline in the blank group, and the two groups were simultaneously physiological. Saline was given to the stomach, 0.2m L/ times per day for 7 days. The mice in each group were selected for eighth days to determine whether the mice had psoriasis like skin lesions by naked eye observation and pathological examination. Then based on the UPLC/Q-TOF MS metabolomics technology, the model group and the blank group of mice plasma samples were metabologically analyzed in order to find the mice psoriasis model and the ordinary. The potential biomarkers.4.MTX of psoriasis patients and the effect of Tripterygium wilfordii polysaccharide on the skin lesions and matching markers of psoriasis mice were used as experimental subjects in 24 female BALB/c mice. After adaptive feeding 3D, 4% chloral chloral (0.1ml/10g) was intraperitoneally injected, and the back hair was removed to form about 2cm * 3cm size. The exposed area was randomly divided into 2 groups, 12 in each group. The group MTX and the group.MTX of the Tripterygium wilfordii group were smeared with 5%IMQ cream 62.5mg on the bare back of the mice daily, while MTX (1mg/kg/d) was administered to the stomach for 7 days. The Tripterygium wilfordii group was smeared with 5%IMQ cream 62.5mg on the bare back of the mice daily and gavage with Tripterygium wilfordii polysaccharide (10mg/kg/d) for 7 days. The mice were harvested at eighth days to determine whether the psoriasis like skin lesions were improved by the naked eye, and then based on the UPLC/Q-TOF MS metabolomics technique, the effects of MTX and Tripterygium wilfordii polysaccharide on the matched markers in psoriasis mice were investigated. The results were: 1. the study of plasma metabolic groups based on the two techniques of ~1H-NMR and UPLC/Q-TOF MS The two techniques of ~1H-NMR and UPLC/Q-TOF MS were used to study the metabolism of plasma in patients with psoriasis vulgaris and healthy volunteers. The results showed that the two groups were distinctions, and there were significant differences in the content of endogenous metabolites between groups. 21 differences were found through ~1H-NMR metabonomics and multivariate statistical analysis. Sexual metabolites, such as very low density lipoprotein (VLDL), low density lipoprotein (low density lipoprotein, LDL), lipids, leucine, isoleucine, valine, beta hydroxybutyric acid, lactic acid, alanine, arginine, N- acetoprotein, acetoacetic acid, acetoacetic acid, pyruvic acid, glutamine, citric acid, creatine, creatinine, Glucose, aspartic acid and phenylalanine. 19 different metabolites were found by UPLC/Q-TOF MS metabonomics and multivariate statistical analysis. They were threonine, leucine, phenylalanine, tryptophan, palmitate amide, linoleate, oleic amide, stearic acid amide, CIS -11- twenty alkenamide, trans -13- twenty-two ene Amides, uric acid, lysophosphatidyl choline (lyso-phosphatidylcholine, Lyso PC) (16:0), Lyso PC (18: 3), Lyso PC (18:2), Lyso PC (18:1), oleic acid, peanut four enoic acid and oleoyl taurine. It is shown from the above results that all two metabolomics techniques have a good clustering effect, but the difference metabolites obtained. The establishment of the model of psoriasis in most different.2.IMQ induced mice and the matching model of the biomarker in the model group and the patient's biomarker can be seen by the naked eye for eighth days. It can be seen that the skin on the back of the blank group of mice is smooth, the skin of the model group of mice is red and the scales are obvious, and the skin skin is thicker. The skin layer of the white group was very thin and the cell morphology was normal. The skin of the model group was hyperkeratosis, keratinization, spinous layer thickening, and a large number of inflammatory cells infiltrated and histopathologically, similar to psoriasis like skin lesions. The model group and blank with UPLC/Q-TOF MS metabolomics technology were used. The metabolomics analysis of the mice plasma samples found that threonine, phenylalanine, tryptophan, palmitate, oleic amide, oleic acid, stearamide, trans -13- twenty-two enamide, Lyso PC (16:0), Lyso PC (18:3), Lyso PC (18:2), and Lyso PC were associated with psoriasis vulgaris in psoriatic mice. Biomarkers are matched. From the above results, we can see that the biomarkers of psoriasis and psoriasis vulgaris have a certain similarity.3.MTX and the effects of Tripterygium wilfordii polysaccharide on the skin lesions and matching markers of psoriasis mice and the eighth days by the naked eye observation, the MTX group and the Tripterygium wilfordii group The erythema, scaly and thickening of the mouse's back skin were improved compared with the model group, indicating that the drug could obviously improve the skin lesions of the mice with psoriasis. The endogenous metabolites of the blank group, the model group, the MTX group and the Tripterygium wilfordii group were studied by the UPLC/Q-TOF MS technique and the multivariate statistical analysis. In the biomarkers matched with the patients, the content of phenylalanine, Lyso PC (16:0), Lyso PC (18:2) and Lyso PC (18:0) in the MTX group decreased, compared with the model mice (P0.05), but there was no statistical difference compared with the blank group. Different (P? 0.05); the content of phenylalanine, tryptophan, Lyso PC (16:0), Lyso PC (18:2) and Lyso PC (18:0) in the group of Tripterygium wilfordii decreased, compared with the model mice (P0.05), but there was no statistical difference (P? 0.05) compared with the blank group (P? 0.05). Amino acids and the level of LPCs. Conclusions: ~1H-NMR and UPLC/Q-TOF MS are two commonly used methods in metabolomics research, and there are some complementarities in biomarker exploration. This article uses two techniques of ~1H-NMR and UPLC/Q-TOF MS to develop endogenous metabolites in patients with psoriasis vulgaris and healthy volunteers. The biological markers found through metabonomics and multivariate statistical analysis will contribute to the diagnosis of disease, the in-depth study of the pathogenesis and the formulation of individualized regimen. The use of IMQ can successfully induce psoriasis like skin lesions in mice and further use the UPLC/Q-TOF MS metabolic group. The effects of MTX and Tripterygium wilfordii polysaccharide on the matched markers in psoriasis mice showed that the therapeutic effect of MTX and Tripterygium wilfordii may be related to the effects of amino acids and the level of LPCs, which will help to establish the pharmacodynamic indexes and provide the basis for the adjustment of drug dosage and individualized administration in the later period.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

【参考文献】