本文選題：尋常型銀屑病 + 代謝組學�。� 參考：《暨南大學》2017年碩士論文

【摘要】：研究背景:尋常型銀屑病是一種常見易復發(fā)的慢性炎癥性皮膚病,目前臨床上對于該病的診斷主要是根據(jù)患者的臨床表現(xiàn)、發(fā)病部位以及組織病理學變化等。但是組織病理學檢查具有一定的創(chuàng)傷性,實驗室常規(guī)檢查指標對尋常型銀屑病的特異性和敏感度較低,臨床價值和意義有限,因此臨床上尚缺少特異性強的尋常型銀屑病診斷標準。此外,該病的病因復雜,發(fā)病機制尚未完全明確,患者常伴有多種內(nèi)源性代謝紊亂。而代謝組學是通過考察生物體系受刺激或擾動后(例如某個基因變異或某一病理生理狀態(tài)下)代謝產(chǎn)物圖譜及其動態(tài)變化來研究生物體系的代謝網(wǎng)絡的一種技術(shù),近年來已經(jīng)在疾病診斷、發(fā)病機制研究、個體化給藥以及藥物開發(fā)等領域得到了廣泛的應用。因此應用代謝組學技術(shù)對尋常型銀屑病患者內(nèi)源性代謝物進行研究并結(jié)合多元統(tǒng)計分析進行模式識別,得到的生物標志物將有助于疾病的診斷、發(fā)病機制的深入研究以及個體化給藥方案的制定。甲氨蝶呤(methotrexate,MTX)和雷公藤多苷是用于銀屑病治療的常用藥物,主要通過免疫抑制來發(fā)揮治療作用,但是其副作用明顯以及個體化差異較大。尋常型銀屑病是一種內(nèi)源性代謝物發(fā)生紊亂的疾病,通過觀察MTX和雷公藤多苷對體內(nèi)生物標志物的影響,可以從代謝物角度出發(fā)來研究其用于銀屑病治療的作用,這將有助于藥效學指標的建立,從而為后期藥物劑量的調(diào)整以及個體化給藥提供依據(jù)。研究目的:1.基于代謝組學技術(shù)探討尋常型銀屑病的生物學標志物。2.探討MTX和雷公藤多苷對銀屑病小鼠與患者相匹配標志物的影響。研究方法:1.樣品的收集與處理選取在暨南大學附屬第一醫(yī)院皮膚科治療且符合納入標準的尋常型銀屑病患者為病例組,以體檢中心的健康志愿者為對照組。清晨空腹抽取患者和健康志愿者的血液樣本,置于5 m L EDTA-K2抗凝管中,采血后立即輕輕顛倒混勻5~6次,靜置2h,離心(4000 r/min,15min,4℃),取上清液于-80℃冰箱凍存待測。2.基于~1H-NMR和UPLC/Q-TOF MS兩種技術(shù)的血漿代謝組學研究分別采用核磁共振(nuclear magnetic resonance ~1H spectroscopy,~1H-NMR)和超高效液相色譜串聯(lián)四級桿飛行時間質(zhì)譜儀(ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry,UPLC/Q-TOF MS)兩種技術(shù)對尋常型銀屑病患者和健康志愿者的血漿樣本進行代謝組學研究,同時結(jié)合主成分分析(principal component analysis,PCA)和正交偏最小二乘法判別分析(orthogonal partial least squares discriminant analysis,OPLS-DA)進行模式識別,對OPLS-DA模型中提取的變量重要性投影(Variable Importance in Projection,VIP)值1的變量進行獨立樣本t檢驗,p0.05為差異有統(tǒng)計學意義,從而篩選出潛在的生物標志物并進行相關代謝途徑分析。然后對這兩種分析方法的結(jié)果進行比較,找到兩者潛在生物標志物中的共性以及特異性指標。3.IMQ誘導小鼠銀屑病模型的建立以及模型組與患者生物標志物的匹配將24只雌性BALB/c小鼠做為實驗對象,適應性喂養(yǎng)3d后用4%水合氯醛(0.1ml/10g)腹腔注射麻醉,剔除其背部毛發(fā),形成約2cm×3cm大小的暴露區(qū)域,隨機分為2組,每組12只,分別為模型組和空白組。模型組每日在小鼠裸露背部涂抹5%咪喹莫特(imiquimod,IMQ)乳膏62.5mg,空白組涂抹等量凡士林,兩組同時用生理鹽水灌胃,0.2m L/次/天,連續(xù)7天。各組小鼠于第8天取材,通過肉眼觀察以及病理學檢查判斷小鼠是否出現(xiàn)銀屑病樣皮損。然后基于UPLC/Q-TOF MS代謝組學技術(shù)對模型組和空白組小鼠血漿樣本進行代謝組學分析,尋找小鼠銀屑病模型中與尋常型銀屑病患者相匹配的潛在生物標志物。4.MTX和雷公藤多苷對銀屑病小鼠皮損部位以及相匹配標志物的影響將24只雌性BALB/c小鼠做為實驗對象,適應性喂養(yǎng)3d后用4%水合氯醛(0.1ml/10g)腹腔注射麻醉,剔除其背部毛發(fā),形成約2cm×3cm大小的暴露區(qū)域,隨機分為2組,每組12只,分別為MTX組和雷公藤多苷組。MTX組每日在小鼠裸露背部涂抹5%IMQ乳膏62.5mg,同時以MTX(1mg/kg/d)灌胃,連續(xù)7天。雷公藤多苷組每日在小鼠裸露背部涂抹5%IMQ乳膏62.5mg,同時以雷公藤多苷(10mg/kg/d)灌胃,連續(xù)7天。各組小鼠于第8天取材,通過肉眼初步判斷小鼠銀屑病樣皮損是否改善,然后基于UPLC/Q-TOF MS代謝組學技術(shù)探討MTX和雷公藤多苷對銀屑病小鼠中與患者相匹配標志物的影響。研究結(jié)果:1.基于~1H-NMR和UPLC/Q-TOF MS兩種技術(shù)的血漿代謝組學研究分別采用~1H-NMR和UPLC/Q-TOF MS兩種技術(shù)對尋常型銀屑病患者和健康志愿者血漿進行代謝組學研究,結(jié)果顯示兩組能夠被明顯區(qū)分,組間內(nèi)源性代謝物的含量存在明顯差異。通過~1H-NMR代謝組學技術(shù)結(jié)合多元統(tǒng)計分析方法找到了21種差異性代謝物,分別為極低密度脂蛋白(very low density lipoprotein,VLDL)、低密度脂蛋白(low density lipoprotein,LDL)、脂類、亮氨酸、異亮氨酸、纈氨酸、β-羥基丁酸、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、檸檬酸、肌酸、肌酐、葡萄糖、天冬氨酸和苯丙氨酸。通過UPLC/Q-TOF MS代謝組學技術(shù)結(jié)合多元統(tǒng)計分析方法找到了19種差異性代謝物,分別為蘇氨酸、亮氨酸、苯丙氨酸、色氨酸、棕櫚酸酰胺、亞油酸酰胺、油酸酰胺、硬脂酸酰胺、順式-11-二十烯酰胺、反式-13-二十二烯酰胺、尿酸、溶血磷脂酰膽堿(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-亞油�；；撬帷囊陨辖Y(jié)果可以看出,兩種代謝組學技術(shù)都具有很好的聚類效果,但是得到的差異性代謝物大部分不同。2.IMQ誘導小鼠銀屑病模型的建立以及模型組與患者生物標志物的匹配造模第8天通過肉眼觀察可以看出,空白組小鼠背部皮膚表面光滑,模型組小鼠背部皮膚紅斑、鱗屑現(xiàn)象明顯,皮膚增厚嚴重。通過病理學檢查可以看出,空白組小鼠皮膚表皮層很薄,細胞形態(tài)正常,模型組小鼠皮膚出現(xiàn)角化過度、角化不全、棘層增厚、皮突延長以及大量的炎性細胞浸潤等組織病理學改變,類似銀屑病樣皮損,初步顯示造模成功。采用UPLC/Q-TOF MS代謝組學技術(shù)對模型組和空白組小鼠血漿樣本進行代謝組學分析發(fā)現(xiàn),在銀屑病小鼠的潛在生物標志物中,蘇氨酸、苯丙氨酸、色氨酸、棕櫚酸酰胺、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)是與尋常型銀屑病患者的生物標志物相匹配的。從以上結(jié)果可以看出,銀屑病小鼠與尋常型銀屑病患者的生物標志物具有一定的相似性。3.MTX和雷公藤多苷對銀屑病小鼠皮損部位以及相匹配標志物的影響給藥第8天通過肉眼觀察可以看出,MTX組和雷公藤多苷組小鼠背部皮膚的紅斑、鱗屑以及增厚現(xiàn)象與模型組相比均有所改善,表明藥物能明顯改善銀屑病小鼠的皮損癥狀。進一步采用UPLC/Q-TOF MS技術(shù)結(jié)合多元統(tǒng)計分析對空白組、模型組、MTX組和雷公藤多苷組的內(nèi)源性代謝物進行研究發(fā)現(xiàn),給藥組與模型組能夠明顯區(qū)分,并有向空白組偏移的趨勢。在銀屑病小鼠與患者相匹配的生物標志物中,MTX組中的苯丙氨酸、Lyso PC(16:0)、Lyso PC(18:2)和Lyso PC(18:0)含量降低,與模型組小鼠相比有統(tǒng)計學差異(P0.05),而與空白組小鼠相比無統(tǒng)計學差異(P㧐0.05);雷公藤多苷組中的苯丙氨酸、色氨酸、Lyso PC(16:0)、Lyso PC(18:2)和Lyso PC(18:0)含量降低,與模型組小鼠相比有統(tǒng)計學差異(P0.05),而與空白組小鼠相比無統(tǒng)計學差異(P㧐0.05)。從以上結(jié)果可以看出,MTX與雷公藤多苷的治療作用可能與影響氨基酸以及LPCs的水平有關。研究結(jié)論:~1H-NMR和UPLC/Q-TOF MS技術(shù)做為代謝組學研究中的兩種常用方法,在生物標志物發(fā)掘中存在一定的互補性。本文采用~1H-NMR和UPLC/Q-TOF MS兩種技術(shù)對尋常型銀屑病患者以及健康志愿者的內(nèi)源性代謝物進行研究都具有很好的聚類效果。通過代謝組學技術(shù)結(jié)合多元統(tǒng)計分析找到的生物標志物將有助于疾病的診斷、發(fā)病機制的深入研究以及個體化給藥方案的制定。采用IMQ可以成功誘導小鼠產(chǎn)生銀屑病樣皮損,進一步采用UPLC/Q-TOF MS代謝組學技術(shù)探討MTX和雷公藤多苷對銀屑病小鼠中與患者相匹配標志物的影響時發(fā)現(xiàn),MTX與雷公藤多苷的治療作用可能與影響氨基酸以及LPCs的水平有關。這將有助于藥效學指標的建立,從而為后期藥物劑量的調(diào)整以及個體化給藥提供依據(jù)。
[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.

【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96

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相關期刊論文 前10條

1 初金玉;劉麗英;鄂佳;楊靜;王晶;;銀屑膠囊聯(lián)合復方氟米松軟膏治療尋常型銀屑病的臨床觀察[J];中國藥房;2016年26期

2 阮智通;底婷婷;王燕;趙京霞;解欣然;王明星;謝湘江;蒙玉嬌;李萍;;養(yǎng)血解毒湯對咪喹莫特誘導的STAT3轉(zhuǎn)基因小鼠銀屑病樣皮損的干預作用[J];中國病理生理雜志;2016年06期

3 謝汶芳;席建元;李小鵬;彭丹;溫柔;;銀屑平丸對BALB/c小鼠銀屑病樣模型血清中IL-17、IL-23表達的影響[J];湖南中醫(yī)藥大學學報;2016年05期

4 劉秀卿;李卓成;吳文中;;雷公藤多苷對咪喹莫特誘導銀屑病樣小鼠Wnt/Frizzled信號傳導通路的影響[J];海南醫(yī)學院學報;2016年11期

5 陳勤;祝驥;盧德趙;;電針對佐劑關節(jié)炎大鼠血漿~1H-NMR代謝譜圖的影響[J];浙江中醫(yī)雜志;2016年02期

6 麥提喀斯木·尼扎木丁;麥合蘇木·艾克木;買吾拉尼江·依孜布拉;維妮拉·烏斯曼;玉蘇甫江·臺外庫力;阿不都熱依木·玉蘇甫;;基于NMR代謝組學方法研究異常黑膽質(zhì)成熟劑對抑郁癥大鼠血漿代謝的影響[J];新疆醫(yī)科大學學報;2016年01期

7 王蓮;夏登梅;李薇;;銀屑病樣動物模型的研究進展[J];中國皮膚性病學雜志;2015年09期

8 熱比姑麗·伊斯拉木;阿瓦姑力·伊斯拉木;斯拉甫·艾白;;銀屑病動物模型研究概況[J];中國藥房;2015年19期

9 高瑤;林東紅;陳敏;歐啟水;杭緯;;基于液相色譜/電噴霧飛行時間質(zhì)譜技術(shù)的結(jié)石癥尿液代謝組學探討[J];分析試驗室;2015年06期

10 馬軍宏;侯艷婷;楊秀竹;于向陽;周振理;;基于代謝組學的膿毒癥實驗研究進展[J];中國中西醫(yī)結(jié)合外科雜志;2014年06期

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