培土清心颗粒对特应性皮炎样小鼠模型的免疫干预研究

发布时间：2017-12-28 06:03

本文关键词：培土清心颗粒对特应性皮炎样小鼠模型的免疫干预研究　出处：《广州中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：本研究为广东省中医药强省建设专项中医优势病种突破项目(粤财社[2015]19号)、特应性皮炎临床和基础研究(粤财社[2011]572号)中的部分研究内容。课题组前期开发研制了具有健脾益气、抗炎止痒的培土清心颗粒(PTQX颗粒),初步建立具有明显皮损的小鼠模型,并验证了其在特应性皮炎(AD)中的临床疗效。本研究拟在前期研究基础上构建AD样小鼠模型和相关评价指标,观察PTQX颗粒对AD样小鼠模型免疫网络系统的影响。研究主要分为2部分。第一部分动物模型构建及评价体系的建立目的：参考国内外文献中特应性皮炎相关动物模型的造模方法,建立特应性皮炎样动物模型及相关评价体系。方法：1.以“特应性皮炎”、“AD”、“动物模型”等为检索词,查找相关文献数据库,根据文献质量评价进行数据提取和分析,参考国内外特应性皮炎动物模型中动物种属、性别选择及具体模型构建方法,筛选适宜的实验动物种属、动物性别和造模方法；2.模型建立后,综合迟发性超敏反应及慢性湿疹评价指标,采用皮损评分、行为观察记分、搔抓次数、耳廓厚度、皮肤病理、血清学等指标评价模型的病症特点。结果：1.国内外特应性皮炎样动物模型研究中,建立AD样动物模型的研究对象宜选用雌性C57BL/6或BALB/c小鼠,采用半抗原刺激诱导法造模,诱导药物采用2,4-硝基氟苯(DNFB)；2.与正常对照组比较,模型组小鼠背部皮损病理表现为明显角化过度、角化不全,棘层肥厚,表皮层、真皮浅层见明显炎性细胞浸润；血清中IgE、IL-4含量均明显升高(P0.01),而IFN-γ未见明显变化(P0.05)。3.造模结束后,模型组小鼠皮损评分、行为观察积分、搔抓次数、耳廓厚度等观察指标与正常组比较,差异均具有统计学意义(P0.05)。其中行为观察记分呈先升后降趋势,与同时点正常组比较,差异有统计学意义(P0.05)；模型组小鼠搔抓次数明显上升、皮损评分、耳廓厚度显著增加(P0.01)。结论：1.采用DNFB诱导法可成功建立AD样小鼠模型,所建立的AD样模型皮肤组织呈较典型慢性改变,血清学表现提示皮肤炎症表现以Th2型细胞为主；2.造模后模型组小鼠显示出较明显的慢性炎症性皮损,伴有瘙痒,与人类AD非常相像,其中皮损记分、瘙痒程度、耳廓厚度、组织病理及血清学改变等可作为判断该模型成功与否的重要依据。第二部分PTQX颗粒对AD样小鼠的免疫调控机制研究目的：通过PTQX颗粒干预AD样小鼠模型,从模型构建影响、血清和皮损细胞因子变化、脾脏Breg细胞数量变化等方面探讨其影响AD免疫调控的可能机制。方法：1.将雌性C57BL/6小鼠随机分为4组,即正常组、模型组、PTQX高剂量组(临床2倍剂量,PTQX颗粒1.56g/kg·d)、PTQX低剂量组(临床等效剂量,PTQX颗粒0.78g/kg·d)。各组小鼠给药方式是灌胃,培土清心用药组用不同浓度水溶液灌胃,正常组和模型组同法给予等体积的蒸馏水,造模第一天开始用药,连续给药14天；实验第1天、第7天、第14天观察各组小鼠行为观察记分、搔抓次数、皮损评分、耳廓厚度等指标；2.采用DNFB反复刺激诱导方法造模,造模前备皮(大小约2.5cm×1.5cm)。造模第一天背部外涂0.5%DNFB溶液25μl致敏,其后第5天、第8天、第11天、第14天在小鼠右耳背面、背部外涂0.2%的DNFB溶液20μl诱发皮炎,左耳外涂基质溶液20μl作对照组；实验结束48小时内予眼球摘除法处死,取背部皮损处全层皮肤做皮肤常规病理和肥大细胞计数变化(甲苯胺蓝染色)；3.采用酶联免疫吸附实验(ELISA)检测小鼠血清中IgE的含量,液相芯片法测定血清中IFN-γ、IL-4、IL-10的含量；4.提取小鼠背部皮肤组织中总RNA,并鉴定其浓度和纯度,采用定量即时聚合酶链式反应(Q-PCR)扩增后检测其皮肤组织中IFN-γ、IL-4、IL-10 mRNA表达水平；5.取小鼠脾脏,分离并培养脾脏淋巴细胞,处理后采用流式细胞术检测脾脏细胞中调节性B细胞(Breg)水平数量及变化。结果：1.造模结束后,模型组小鼠搔抓次数、皮损评分、耳廓厚度较正常组明显升高(P0.01),给予PTQX颗粒后,小鼠搔抓次数、皮损评分、耳廓厚度均明显降低(P0.01)；行为观察记分趋向于正常,与正常组比较差异无统计学意义(P0.05)；皮肤组织病理棘层肥厚较模型组减轻,皮肤炎性细胞浸润减少,肥大细胞计数明显降低(P0.01)；2.与正常对照组比较,模型组小鼠血清IgE、IL-4含量增加(P0.01),血清IFN-γ、IL-10未见明显改变(P0.05),给予PTQX颗粒后,PTQX高剂量组与PTQX低剂量组小鼠血清中IgE含量均明显降低(P0.05),PTQX低剂量组小鼠血清中IL-4含量与模型组比较,差异具有统计学意义(P0.05),PTQX高剂量组血清中IL-4含量与模型组比较呈下降趋势;两组用药组小鼠血清中IFN-γ、IL-10含量与模型组比较,差异均无统计学意义(P0.05)；3.Q-PCR结果显示造模后,小鼠皮肤组织的i110 mRNA、ifnγ mRNA、i14 mRNA表达量均上升。给予PTQX颗粒后,不同剂量用药组ifnγ mRNA、il4 mRNA表达量均减少,与模型组比较,差异均具有统计学意义(P0.05),而小鼠皮肤组织i110mRNA表达量在PTQX颗粒干预后明显升高,与模型组比较差异明显(P0.05)。4.造模后,小鼠脾脏内CD5+B淋巴细胞整体数量所占淋巴细胞的比率明显下降(P0.01),给予PTQX颗粒后,PTQX高剂量组和PTQX低剂量组CD5+B淋巴细胞较模型组明显升高(P0.05),趋向于正常。而造模后小鼠脾脏内CD4+T淋巴细胞、CD4+IL-4+T淋巴细胞、CD4+IFN-γ+T淋巴细胞、CD4+IL-17+T淋巴细胞、CD5+CD19+淋巴细胞、CD5+CDld+B淋巴细胞、Breg细胞阳性表达率均未见明显改变,给予PTQX颗粒后,PTQX高剂量组CD5+CD1d+B淋巴细胞阳性表达率明显增多(P0.05),而低剂量组呈增多趋势(P0.05)；PTQX高剂量组Breg细胞阳性表达率明显升高,与模型组比较,差异具有统计学意义(P0.05),而低剂量组呈增多趋势(P0.05),其余淋巴细胞阳性表达率与与模型组比较,差异也均无统计学意义(P0.05)。结论：1.PTQX颗粒可有效改善AD样小鼠模型皮损情况,减轻瘙痒,减少皮肤炎性细胞浸润；2.IgE、IFN-γ、IL-4、IL-10是培土清心颗粒干预AD的重要靶点,PTQX颗粒治疗AD可能与下调血清IgE、IL-4含量和皮肤中ifnγ mRNA、il4mRNA含量,上调i110mRNA含量有关,通过纠正上述细胞因子紊乱、恢复体内免疫稳态而发挥治疗作用；3.PTQX颗粒可能通过刺激CD5‘B淋巴细胞、CD5+CDld+B淋巴细胞、Breg细胞增殖,调节特应性皮炎患者免疫失衡状态。
[Abstract]:This study is a part of the research on the special Chinese medicine dominant disease breakthrough project (Guangdong Finance Association [2015]19), the clinical and basic research of atopic dermatitis (Yuecai [2011]572) in Guangdong province. Our research group developed particles with Spleen Qi, heart Peitu anti-inflammatory antipruritic (PTQX particles), the initial establishment of a mouse model with obvious lesions, and validated in atopic dermatitis (AD) clinical curative effect in. The aim of this study is to construct AD - like mouse model and related evaluation index on the basis of previous studies, and to observe the effect of PTQX particles on the immune network system of AD like mice. The research is divided into 2 parts. The first part is the establishment of animal models and the establishment of evaluation system: Objective: to establish the atopic dermatitis related animal models and related evaluation system based on the modeling methods of atopic dermatitis related animal models at home and abroad. Methods: 1. "atopic dermatitis", "AD", "animal model" as the key words, find relevant literature database, data extraction and analysis according to the quality evaluation of literature, reference to domestic and foreign atopic dermatitis animal model in animal species, sex selection and specific model construction method, suitable for screening the experimental animal species, sex and modeling methods; 2. after the establishment of the model, comprehensive delayed hypersensitivity and chronic eczema evaluation index, the lesion score and behavior observation score, scratching number, ear thickness, skin pathology, serum and index evaluation models of disease characteristics. Results: 1. of the domestic and foreign research on atopic dermatitis like animal model, the research object to establish AD like animal model should be selected female C57BL/6 or BALB/c mice by semi antigen stimulation induced model induced by 2,4-, drug 4-dinitrofluorobenzene (DNFB; 2.) compared with the normal control group, model group mice back pathological lesions obvious hyperkeratosis, parakeratosis, acanthosis, epidermis, dermis showed obvious inflammatory cell infiltration; serum IgE, IL-4 levels were significantly increased (P0.01), and IFN- gamma showed no significant change (P0.05). 3. after modeling, there were significant differences in skin lesions score, behavioral observation score, scratch times and ear thickness between model group and normal group (P0.05). Among them, behavior observation score increased first and then decreased. Compared with the normal group at the same time, the difference was statistically significant (P0.05). The number of scratch times in the model group increased significantly, and the score of skin lesions and the thickness of the auricle increased significantly (P0.01). Conclusion: 1. induced by DNFB method can successfully establish AD mouse model, AD model of skin tissue established a typical chronic inflammation of the skin changes, serological manifestations suggestive in type Th2 cells; 2. model group mice showed chronic inflammatory skin damage, obviously accompanied by itching, and human AD is very similar, the lesion score, pruritus degree, ear thickness, histological and serological changes can be used as an important basis for judging the success of the model. Study on the immune regulation mechanism to the second part of PTQX particles on AD like mice by PTQX Granule on AD like mouse model, from the model, effects of change, blood and skin cell factor number of spleen Breg cells change and to explore the possible mechanism of immune regulation of AD. Methods: 1.. The female C57BL/6 mice were randomly divided into 4 groups, namely, normal group, model group, PTQX high dose group (2 times clinical dose, PTQX granule 1.56g/kg d), PTQX low dose group (clinical equivalent dose, PTQX granule 0.78g/kg. D). Mice were administered orally, medication group hilling and clearing away heart fire with water solution of different concentration by gavage, the normal group and model group were given equal volume of distilled water, the first day of modeling medication administered continuously for 14 days; the first day, seventh day and fourteenth day were observed behavior observation score, scratching number and lesion score, ear thickness and other indicators; 2. by repeated DNFB stimulation induced modeling method, before modeling skin (about the size of 2.5cm * 1.5cm). Modeling the first day back with 0.5%DNFB solution 25 l sensitization, 20 l induced dermatitis DNFB solution within the next fifth days, eighth days, eleventh days, fourteenth days in the mouse ear back, back with 0.2% of the left ear with the matrix solution 20 l for the control group given within 48 hours of the end of the experiment; the method of enucleation were taken back full-thickness skin lesions do change skin routine pathology and mast cell count (toluidine blue staining); 3. by enzyme-linked immunosorbent assay (ELISA) to detect the content of serum IgE content, serum IFN-, IL-4, IL-10 y Liquichip; RNA 4. from the back skin of mice, and identify its concentration and purity, using quantitative real-time polymerase chain reaction (Q-PCR), IL-4, IL-10 level IFN- gamma mRNA expression in the skin tissue was examined; 5. mice spleen, isolated and cultured spleen cells, after treatment The number and changes of regulatory B cells (Breg) in spleen cells were detected by flow cytometry. Results: 1. weeks after the modeling, model group mice scratching frequency, lesion score and auricle thickness increased significantly compared with normal group (P0.01), PTQX particles, mouse scratching number, lesion score and auricle thickness were significantly decreased (P0.01); behavior observation score tends to Yu Zheng Chang, no statistical significance compared with the normal the difference (P0.05); skin tissue pathological acanthosis alleviated as compared with model group, reduce skin infiltration of inflammatory cells and mast cell count decreased significantly (P0.01; 2.) compared with the normal control group, model group, serum IgE, IL-4 content increased (P0.01), serum IFN- gamma, IL-10 did not change significantly (P0.05), PTQX particles, PTQX high dose group and low dose group of PTQX mice serum IgE levels were significantly decreased (P0.05) compared with the model group, the content of IL-4 in serum of mice PTQX low dose group, the difference was statistically significant (P0.05, PTQX)
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285.5;R-332

【相似文献】