【作者】 周爱妍；

【导师】 杜锡贤；

【作者基本信息】 山东中医药大学 ， 中医外科学， 2013， 博士

【摘要】 研究背景：特应性皮炎(AD)是一种慢性、复发性、变态反应性皮肤病，属于中医文献中的“奶癣”、“四弯风”、“湿疮”、“浸淫疮”等范畴。脾虚湿困是其主要病机。诸多医家采用健脾化湿法治疗AD取得了良好的临床疗效，已报导的作用机理主要包括抗炎及免疫调节方面。水通道蛋白（AQPs）在机体的多个组织器官表达，在维持体内水平衡过程中起重要作用。目前诸多研究已证明水通道蛋白与水液代谢障碍性疾病有密切关系，与中医津液代谢理论相关。深入研究水通道蛋白的作用，将有可能解释水、湿等病理变化的机理，并在此基础上阐明化湿、渗湿、燥湿药的药理作用。已有研究表明水通道蛋白3（AQP3）与AD发病相关。健脾化湿法是否能够通过调节皮肤AQP3来发挥治疗作用还存在许多空白。本研究运用健脾化湿法治疗AD小鼠，运用现代分子生物学研究技术观察健脾化湿法对AD小鼠皮肤AQP3表达的影响，旨在探讨健脾化湿法治疗AD的部分作用机制与途径，为进一步探明健脾化湿法治疗AD的分子生物学机制奠定基础。研究目的：1、初步探讨导师小儿健脾化湿方治疗AD患儿的疗效，为指导临床治疗提供依据。2、通过观察健脾化湿法对AD小鼠AQP3表达的影响，初步探讨健脾化湿法治疗AD的部分作用机制与途径，为进一步阐明健脾化湿法治疗AD分子生物学机制奠定基础。研究方法：1、本研究选择50例脾虚湿蕴型AD患儿为研究对象，治疗前及治疗后4周时分别记录SCORAD积分的改善。治疗前及治疗4周后检测血清总IgE和EOS计数。2、采用免疫组织化学法检测AD小鼠皮肤组织AQP3的表达变化。3、应用荧光定量PCR法、Western blot法检测小儿健脾化湿方对AD小鼠皮肤组织AQP3mRNA及蛋白的影响。研究结果：1、25例AD患儿经小儿健脾化湿方治疗4周后SCORAD积分下降明显，有显著性差异(P＜0.01)，说明健脾化湿法治疗儿童AD疗效确切。2、血清总IgE的测定：治疗组治疗前血清总IgE较健康对照组显著升高，差异有统计学意义(P＜0.01)；治疗后治疗组血清总IgE较治疗前降低，差异有统计学意义(P＜0.01)。对照组治疗前血清总IgE较健康对照组显著升高，差异有统计学意义(P＜0.01)；治疗后对照组血清总IgE与治疗前比较无明显差异（P＞0.05）。3、嗜酸性粒细胞测定：治疗组治疗前EOS较健康对照组显著升高，差异有统计学意义(P＜0.01)；治疗后治疗组EOS较治疗前降低，差异有统计学意义(P＜0.01)。对照组治疗前EOS较健康对照组显著升高，差异有统计学意义(P＜0.01)；治疗后对照组EOS与治疗前比较无明显差异（P＞0.05）。4、免疫组织化学法检测发现，AD小鼠皮肤AQP3平均光密度值升高，与空白对照组比较差异有统计学意义(P＜0.01)。中药模型组小鼠皮肤组织AQP3平均光密度较空白模型组降低，其间差异有统计学意义(P＜0.01)。5、Western blot检测结果显示空白模型组小鼠皮肤组织中AQP3蛋白较空白对照组明显升高，差异有统计学意义(P＜0.01)。中药模型组较空白模型组显著降低，差异有统计学意义(P＜0.01)。荧光免疫PCR检测显示空白模型组小鼠皮肤组织中AQP3mRNA表达较空白对照组明显升高，差异有统计学意义(P＜0.01)。中药模型组AQP3mRNA较空白模型组降低，差异有统计学意义(P＜0.01)。结论：1、小儿健脾化湿方治疗AD患儿临床症状改善明显，SCORAD积分显著降低，差异有统计学意义。说明健脾化湿法治疗儿童AD疗效确切。并且在治疗期间没有观察到明显的副反应，初步证实本法治疗AD安全有效，值得推广。2、本研究显示AD患者血清总IgE、EOS水平明显升高，治疗后血清总IgE、EOS水平明显降低，差异有统计学意义，说明此二者均参与了AD的发病，可作为反映AD患者病情严重程度的血清学指标之一。3、AD模型鼠皮肤组织AQP3蛋白及AQP3mRNA表达显著升高，经小儿健脾化湿方治疗后皮肤组织AQP3表达降低，说明健脾化湿法治疗可以明显改善皮肤组织中AQP3蛋白及AQP3mRNA的表达，促进皮肤的修复。更多还原

【Abstract】 Research background:Atopic Dermatitis (AD) is a chronic, recurrent, allergic skin, belonging to theliterature of traditional Chinese medicine "infantile eczema,""four-bend wind","eczema","acute eczema" and other areas. Dempness stagnancy due to spleen deficiency is acommon pathogenesis.Treatment of strengthening spleen and removing dampness has beenused by many doctors,and achieve good clinical efficacy.Mechanisms that have beenreported include anti-inflammatory and immune regulation. Aquaporins (AQPs) expressedin many organizations in body, suggests that they play an important role in maintaining thewater balance in the body. Study the role of water channel protein in depth, is expected tobe a very good explanation of pathological changes such as sputum, drinking water, wet,mechanism, and on this basis formulate dampness medicine, Li water wet, dry wetpharmacological effects of drugs. Many studies have demonstrated that aquaporin closelyrelated to water metabolism disorders and Chinese medicine theory of body fluidmetabolism.Research has indicated that the water channel aquaporin-3associated with thepathogenesis of AD. There are many gaps that if the method of strengthening spleen andremoving dampness can adjust AQP3in thetreatment of the skin or not. In this study, weuse the treatment of strengthening spleen and removing dampness in ad mice. To explorethe mechanism and approach in treatment method of strengthening the spleen andremoving dampness in AD. To further verify to the molecular biology mechanism of thetreatment of strengthening spleen and removing dampness in AD, and lays the foundationfor it. Research purpose:1.Discusses the efficacy of xiao er jian pi hua shi fang in treating children with AD,and provide a basis for treatment in clinical AD.2.By observing the impact of treatment of strengthening spleen and removingdampness to the aqp-3expression on the ad the mice, to further verify to the molecularbiology mechanism of the treatment of strengthening spleen and removing dampness inAD, and lays the foundation for it.Research methods:1.50children with AD for the study, recorde SCORAD score before treatment and4weeks after treatment. detect Serum total IgE and EOS.2. Immunohistochemical detection of atopic dermatitis skin tissues of mice by AQP3expression;3.Using immunohistochemical method, RT-PCR method, Western blot assay methodof jianpi dampness on atopic dermatitis AQP3expression of skin tissues of mice.Research results:1.SCORAD score of25children with AD dropped significantly after4weeks after thetreatment of strengthening spleen and removing dampness. The difference has thestatistical significance(P＜0.01), Showing that the treatment of strengthening spleen andremoving dampness on children’s AD has remarkable curative effect.2. Determination of total serum IgE: Before treatment the blood serum total IgE ofgroup treatment obviously elevates than healthy control group, The difference has thestatistical significance(P＜0.01); After treatment total serum IgE of group treatmen islower than before treatment, The difference has the statistical significance(P＜0.01);Serum total IgE of control group before the treatment is significantly higher compared withhealthy control group, The difference has the statistical significance(P＜0.01); Serum totalIgE of treatment control group has not significantly difference compared with beforetreatment（P＞0.05）.3. Determination of eosinophil cells: Before treatment the EOS of group treatmentobviously elevates than healthy control group, The difference has the statisticalsignificance(P＜0.01); After treatment, the treatment group EOS is lower than beforetreatment, The difference has the statistical significance(P＜0.01); EOS of control groupbefore the treatment is significantly higher compared with healthy control group, Thedifference has the statistical significance(P＜0.01); EOS of treatment control group has not significantly difference compared with before treatment（P＞0.05）.4. Immunohistochemical method detected discovered that verage light density ofAQP3in AD mouse skin obviously elevates, the difference has the statistical significancethan blank control group(P＜0.05); The verage light density of AQP3in chinese medicinemodel mice skin decreased compared with blank model group, the difference has thestatistical significance(P＜0.05).5. Western blot test results: Compared with the blank control group, AQP3protein inthe blank model mice skin significantly elevated, the difference has the statisticalsignificance(P＜0.01); Chinese medicine model group dramatically reduces than blankmodel group, the difference has the statistical significance(P＜0.01). RT-PCR testing:Compared with the blank control group, AQP3mRNA in the blank model mice skinsignificantly elevated, the difference has the statistical significance(P＜0.01); Chinesemedicine model group dramatically reduces than blank model group, the difference has thestatistical significance(P＜0.01).Conclusion:1. Clinical symptoms of AD in patients improved significantly after treatment of xiaoer jian pi hua shi fang, the SCORAD integral dramatically reduces, has statisticalsignificance, showing that the treatment of strengthening spleen and removing dampness inchildren’s AD has curative effect to be accurate.2. This study also confirmed that the majority of the AD in patients with markedlyelevated serum total IgE and EOS, serum total IgE and EOS levels after treatmentsignificantly reduced, statistically significant differences, they involved in the pathogenesisof AD, and reflect the severity of AD.3. Atopic Dermatitis model mice skin tissue AQP3expression significantly higher,after treated with jianpi huashi AQP3expression in skin reduce, so jianpi dampnesstreatment can significantly improve the expression of AQP3mRNA in skin, promote skinrepair.更多还原