【作者】 赵丽萍；

【导师】 高兴华；

【作者基本信息】 中国医科大学 ， 皮肤病与性病学， 2007， 博士

【摘要】 中国北方异位性皮炎的流行病学及与SPINK5、SCCE基因多态性关系的研究前言异位性皮炎（Atopic Dermatitis，AD）的病因不明，影响因素较多。近年来的研究进展已使人们认同：异位性皮炎并非单一的病种，而是具有一定共同特征的若干疾病的集合体。认为AD特征性的临床表型是易感性基因、环境、有缺陷的皮肤屏障功能、免疫学反应间相互作用的产物等。AD具有“异位性”，常继发或伴有哮喘、过敏性鼻炎、枯草热等。AD患者以青少年为多，有10％左右可迁延至成年期，其发病率随着年龄的增长而逐渐下降趋势。经相关分析证明，患病率与年龄呈负相关。全球范围内，AD的患病率分布不均衡。在我国的不同地区，AD的患病率也有不同的特点。近年来，AD的发病率呈现逐渐上升的趋势，尤其以工业发达国家为著，如欧洲、日本等地。AD的病因未知。环境因素在AD发展中有重要的作用。不同地区AD患者中接触变应原的分布有不同的特点。AD与表皮屏障功能障碍有关。AD以皮肤干燥为特征，甚至于非皮损处的皮肤亦受累，经表皮水份丢失增加。AD中表皮屏障功能受损导致过敏原吸收增加，从而导致AD的皮肤高反应性。AD对刺激物的易感性增加，因此在临床上表现出原发性的表皮分化缺陷与炎症诱发的皮肤损害表现共存。遗传学方面的资料显示AD有高度的遗传性。基因学连锁和相关性的研究已测绘出多个AD的疾病位点图，尽管未明确主要的致病基因，但已证实了AD与许多染色体区域连锁，如1q21、3q21、5q31-33、11q13、17q25、20p。在染色体5q31-33区域包含了一串Th2细胞因子基因，如IL-3、4、5、13，GM-CSF及丝氨酸蛋白酶抑制因子5（serine protease inhibitor，Kazal-type，5SPINK5）基因。SPINK5基因编码淋巴上皮Kazal5型LEKTI蛋白，是一种丝氨酸蛋白酶抑制剂。对内塞顿综合征（Netheton sydrome NS）的研究发现，SPINK5基因的突变是导致该病的病因。NS包括临床三联征：先天性干皮病，内折性脆发症及异位表现：如异位皮炎，血管性水肿，荨麻疹，血清IgE升高及EOS增多症。因为AD具有皮肤干燥，异位症，皮肤屏障功能障碍等与NS相似之处，故认为SPINK5基因可能与AD有某种关联。目前于欧洲及日本已有多项研究证实AD与SPINK5基因Glu420Lys多态性相关，但也有不同的结论。角化层糜蛋白酶（stratum corneum chymotryptic enzyme，SCCE）基因是组织激肽释放酶（KLK）基因家族成员之一（KLK7），编码hk7蛋白，特异性地于角质层上皮细胞中表达，是一种丝氨酸蛋白酶。研究发现SCCE主要催化皮肤最外面角化层的细胞间粘连结构的降解，导致皮肤表面脱屑。除了它的组织分布特点外，SCCE有几种与表皮脱屑相关的重要的基因先决条件相匹配的特点，包括PH值与催化活性的抑制剂。高表达人SCCE的转基因鼠的皮肤发生的改变与在慢性AD中所见的特点相似。在最初时，过表达SCCE可能导致表皮屏障过早破坏。在角层的提取物中，SCCE与另一个丝氨酸蛋白酶角层胰蛋白酶（KLK）5一起作为总的蛋白水解活性的主要部分。LEKTI是它的一种抑制剂，丝氨酸蛋白酶与其抑制剂之间的平衡在皮肤脱屑的调节中起中心作用。SCCE在皮肤中表达，与多种皮肤疾病有关。HanssonL等发现KLK7在异位性皮炎等慢性皮肤损害中高表达，可能与炎性皮肤病的发病机理有关。并认为SCCE可作为慢性炎性皮肤病新的治疗潜在靶标。近来有研究表明SCCE基因3’UTR区AACC插入与AD密切相关。但缺乏多次重复的研究来证实。研究对象与方法一、SPINK5基因多态性与异位性皮炎相关性的研究（一）、研究对象1、病例组即异位性皮炎组。为2005年3月至2007年1月在中国医科大学附属第一医院皮肤科门诊就诊的沈阳及周边地区患者91例。诊断标准为williams标准。所有病例于就诊前1个月未内用糖皮质类固醇激素及免疫抑制剂。以化学发光法检测血清IgE水平。2、正常对照组与辽宁省血液中心合作，收集健康志愿者的血液样本。两组男女比例匹配。沈阳地区正常志愿献血者250例。已排除有过敏性哮喘、过敏性鼻炎及荨麻疹、湿疹、枯草热等异位性表现者。血液样品处理：抽静脉血3ml，用2％EDTA抗凝。置于－80℃深低温冰箱中保存，以备提基因组DNA。（二）实验方法：用聚合酶链反应（PCR）—限制性片段长度多态性分析方法（restncuon fragment ieng}n polymorphism RFLP）1、采用酚—氯仿法提取基因组DNA从NCBI数据库中获取SPINK5基因（Genebank NM₀06846）的序列，参照文献设计引物序列。PCR的体系：10×LAbuffer 2ul，上游引物0.5ul，下游引物0.5ul，dNTP 2ul，LATaq酶0.1ul，DNA模板1ul，双蒸水加至20ul。PCR的条件：94℃3min预变性后，经35个循环，每个循环的组成：94℃30s，52℃－53℃30s，72℃30s，最后延伸72℃5min。经PCR方法扩增包含A1103G（Asn368Ser）、G1156A（Asp386Asn）、G1258A（Glu420Lys）、G2475T（Glu825Asp）多态位点的相应片段。扩增的片段大小分别为：A1103G：367bp；G1156A：367 bp；G1258A：356 bp；G2475T∶325 bp。上述四个基因位点分别用限制性内切酶Bts I，Mbo I，Hinf I，FOK I，在37℃水浴10小时进行消化。用2％琼脂糖凝胶电泳分离方法进行分析。2、统计学处理方法所有资料均用统计软件包SPSS11.5建立数据库。记录全部个体的分型结果。所有统计学分析过程均采用SPSS11.5软件完成。年龄以中位数土四分位间距表示。计算各等位基因与基因型频率。等位基因频率和基因型频率采用直接基因计数法计算，等位基因频率=等位基因阳性数／（总个体数×2），基因型频率=携带等位基因的人数／总个体数。Hardy-Weinberg平衡用卡方检验，应用自由度为1的卡方检验来判断各多态位点是否符合Hardy-Weinberg平衡。卡方检验还用于单因素分析时检验多态与疾病间的关联。对病例组的部分相关因素行单因素非条件logistic回归分析。P值小于0.05差异具有显著性。二、SCCE基因多态性与异位性皮炎相关性的研究（一）研究对象1、病例组即异位性皮炎组。2005年3月至2007年1月在中国医科大学附属一院皮肤科门诊就诊的沈阳及周边地区患者60例。诊断标准为williams标准。所有病例于就诊前1个月未内用糖皮质类固醇激素及免疫抑制剂。以化学发光法检测血清IgE水平。2、正常对照组为沈阳地区正常志愿献血者120例。已排除有过敏性哮喘、过敏性鼻炎及荨麻疹、湿疹、枯草热等异位性表现者。与辽宁省血液中心合作，收集健康志愿者的血液样本。两组男女比例匹配。血液样品处理：抽静脉血3ml，用2％EDTA抗凝。置于－80℃深低温冰箱中保存，以备提基因组DNA。（二）实验方法1、用等位基因特异性PCR方法研究样品提取基因组DNA详见论文一。从NCBI数据库中获取SCCE基因（Genebank NM₀05046）的序列，用GENE RUNNER及Dnaman软件设计引物。其中反义链R2的3’末端与突变型的等位基因互补，而反义链R1则于野生型等位基因互补，形成片段长度为363bp。反义链R3与正义链F形成的片段长度为192bp，为内参照片段。PCR扩增：每个样品分别进行2次PCR反应，分别加入反义链R1及R2，将2次PCR反应的结果结合起来分析基因型结果。PCR体系：10×Labuffer1ul，上游引物F 0.5ul，下游引物R1／R2 0.3ul，下游引物R3 0.2，dNTP 2ul，LA Taq酶0.1ul，DNA模板1ul，双蒸水加至20ul。PCR扩增：94℃3min预变性后，经35个循环，每个循环的组成：94℃30s，58℃30s，72℃30s，最后再72℃延伸5分钟。2、统计方法同论文一。三、异位性皮炎流行病学调查的研究（一）研究对象7000例，分别来自市第85中学、第86中学、第90中学、浑南第1小学、沈阳大学机关幼儿园、本溪市实验小学及本溪市第二高中的学生。（二）研究方法1、流行病学调查以问卷方式进行。问卷内容主要包括由Williams等设计、已在我国南京地区中小学生调查中证实具有较高敏感性和特异性的5个问题。此外，尚有一般情况（年龄、性别、家族史、疾病、诱因、居住环境、治疗情况）、问卷中对瘙痒和屈侧等名词作了具体解释。问卷由学校校医以班级为单位统一分发、收集，要求家长如实填写或协助填写。由专业调查员（从事皮肤科临床工作多年的主治医师）对问卷进行统一筛查。2、由调查员到学校校医室对根据问卷内容诊断为AD的调查对象进行抽样专科体检，同时仔细核实问卷内容。用Williams标准确诊异位性皮炎。3、所有资料均用统计软件包SPSS11．5建立数据库，采用双遍输入，进行核对。发现有问题的数据，随即查对原始记录进行校正。所有统计学分析过程均采用SPSS11．5软件完成。年龄以中位数士四分位间距表示。以沈阳及本溪地区人群总和为标准组，用直接法对患病率进行标化。计数资料组间比较采用χ²检验。对影响因素进行了单因素非条件logistic回归分析。α＜0.05。结果一、SPINK5基因多态性与AD的相关研究1、研究对象一般情况AD病例共91人，年龄1岁～67岁，年龄中位数为15岁±19岁。男性1～10岁年龄组的比例高于女性组，而女性组31～67岁年龄组的比例高于男性组。病例组中有39例病人血清IgE水平高于正常值，在不同性别间及不同年龄组间分布无显著性差异。在AD的相关因素（过敏性鼻炎、过敏性哮喘、经常皮肤瘙痒、经常出汗时瘙痒、全身皮肤干燥、身体屈侧皮炎、2岁前发病、初发于面部）中，病例组人群在不同性别组中分布均衡。家族异位症史阳性人群在不同性别组中分布均衡。按血清IgE水平高低不同分组，以正常值最高限165IU／ml为界，小于该数值者为第一组：1～165IU／ml；大于该数值者为第二组：166～3000IU／ml，在两组间年龄分布无显著差别，P＞0.05。2、病例组与对照组比较在本研究中，病例组人群中SPINK5基因位点1103G、1156A、1258A、2475T的等位基因频率分别为：0.60、0.44、0.54、0.60，正常对照组人群SPINK5基因位点1103G、1156A、1258A、2475T等位基因频率分别为：0.47、0.45、0.48、0.47。基因位点2475T多态等位基因频率在两组间差别显著，χ²=4.697，P=0.03。A1103G、G1156A、G1258A位点的等位基因频率在两组间的差异无显著性，P＞0.05。病例组和对照组间基因位点A1103G、G1258A、G2475T的基因型频率差别显著，P值分别为0.000、0.000、0.035。基因位点G1156A的基因型频率在两组间分布差异无显著性，P＞0.05。3、病例组内各相关因素及IgE不同水平组间比较过敏性哮喘、一级亲属有异位症史等因素的分析结果发现：基因位点G2475T中GG及GT基因型频率在AD伴过敏性哮喘病人及AD不伴有过敏性哮喘病人之间有显著统计学差异（P＜0.05）。基因位点G2475T的基因型GT是AD伴有过敏性哮喘的危险因素（OR=7.744，95％C．I∶2.080～28.835）。其它各相关因素的单因素分析无显著统计学差异（P＞0.05）。在不同水平的IgE组间，无显著性的统计学差异（P＞0.05）。二、SCCE基因多态性与AD的相关研究1、研究对象一般情况AD病例60人，年龄1岁～64岁，年龄中位数为14.5岁±14.5岁。男性31人，女性29人，男女比例1.45∶1。在不同性别间及不同年龄组间分布均衡。病例组中有26例病人血清IgE水平高于正常值，在不同性别间及不同年龄组间分布无显著性差异在AD的相关因素（过敏性鼻炎、过敏性哮喘、经常皮肤瘙痒、经常出汗时瘙痒、全身皮肤干燥、身体屈侧皮炎、2岁前发病、初发于面部）中，病例组人群在不同性别组中分布均衡．家族异位症史阳性人群在不同性别组中分布均衡。家族异位症史阳性人群在不同性别组中分布均衡。对照组共120例，男性71人，女性49人，男性比例1.27∶1。年龄19岁～73岁，年龄中位数为27岁±8。从表6中可见病例组及对照组人群在性别间的分布均衡。2、病例组与对照组间比较病例组中基因型AACC／AACC、AACC／AACCAACC、AACCAACC／AACCAACC的基因型频率分别为0.12、0.08、0.78；等位基因AACC、AACCAACC的频率分别为0.17、0.83。对照组中AACC／AACC、AACC／AACCAACC、AACCAACC／AACCAACC的基因型频率分别为0.279、0.287、0.435；等位基因AACC、AACCAACC的频率分别为0.41、0.59。SCCE基因AACC插入多态的等位基因频率及基因型频率在病例组及对照组间差别显著，P=0.000。3、病例组内各相关因素及IgE不同水平组间比较AD相关因素的单因素分析结果发现SCCE基因AACC插入多态等位基因频率在AD病人伴有一级亲属异位症和AD病人不伴有一级亲属异位症间差别显著，χ²=8.143，P=0.006，但经单因素非条件Logistic回归分析无统计意义。其它各相关因素经单因素非条件Logistic回归分析，差异无显著性（P＞0.05）。在不同水平的IgE组间，差异无显著性（P＞0.05）。三、异位性皮炎的流行病学调查1、研究对象一般情况本次调查人口总数7000人，回收问卷6794份。在回收的问卷中，有654份因填写不规范而未被录入数据库进行统计学分析，有效调查问卷为6140份。年龄中位数为15±4岁，年龄分布以6—16岁者为多，而大于或小于这一年龄段者相对偏少。在沈阳地区组及本溪地区组间所调查的人群的年龄分布有显著性差异。男性2661人，女性2482人，沈阳地区男性880人，女性907人，本溪地区男性1781人，女性2482人，在被调查的总人群中，4919为汉族，1221为少数民族，其中以满族、回族、鲜族、蒙古族为主。2、AD的患病率2006年7～9月，两地总的患病率为0.98。男性标化患病率为1.19，女性标化患病率为0.82，差异无显著意义，P＞0.05。经年龄标化后沈阳市标化患病率为0.28％，其中沈阳地区男性标化患病率为0.52％，女性标化患病率，为0.05％，本溪地区标化患病率为1.08％，其中本溪地区男性标化患病率为1.24％，女性标化患病率为0.96％；沈阳本溪两地区间患病率比较没有显著性差异，X²MH=1.325，P=0.250。经性别标化后，沈阳地区的患病率为0.16％，本溪地区的患病率为1.03％，两地患病率比较有显著的差异，X²MH=6.977，P=0.008。沈阳地区的标化患病率显著低于两地总的患病率，χ²=4.499，P=0.040。本溪地区3～12岁组AD的患病率高于13～20岁组，但差异无明显性，P＞0.05。本溪地区的3～12岁组的患病率高于沈阳地区，但差异无显著性，P＞0.05。3、AD的相关因素AD患者伴有皮肤干燥、过敏性鼻炎者、过敏性哮喘、身体屈侧皮炎、经常性的皮肤瘙痒、经常皮肤出汗时瘙痒的伴发率较高。各项因素在男女性别间比较差异无显著性。在AD中患者身体各部位发生皮炎情况，依患病率高至低的部位为：手、关节和躯干、颈部、足部、四肢屈侧、面部、四肢伸。男女性别间患病率各部位比较差异均无显著性。60例AD中，一级亲属中有异位症者有16（26.7％），二级亲属5（8.3％），三级亲属3（5.0％）。经单因素及多因素的非条件logistic回归分析显示AD的影响因素在AD组与非AD组间差异有显著意义（P＜0.05），过敏性鼻炎、过敏性哮喘、身体屈侧皮炎、经常性的皮肤瘙痒、经常皮肤出汗时瘙痒、一级亲属有异位症史者是AD的危险因素。4、可能与AD有关的影响因素60例AD患者中共有34例患者其家长吸烟。AD组与非AD组比较，家长吸烟率没有显著性差异（P＞0.05）。经单因素及多因素非条件Logistic回归分析显示家里潮湿、家里有宠物是AD的危险因素，剖腹产是AD的保护性因素。结论1、SPINK5基因A1103G（Asn368Ser）、G1258A（Glu420Lys）、G2475T（Glu825Asp）多态与AD有密切关系。在有、无AD的相关因素（过敏性鼻炎、过敏性哮喘、经常皮肤瘙痒、经常出汗时瘙痒、全身皮肤干燥、身体屈侧皮炎、2岁前发病、初发于面部）人群中，基因型及等位基因频率均无显著性差别。基因位点G2475T的基因型GT是AD伴有过敏性哮喘的危险因素。2、SCCE基因3’UTR区AACC插入多态性与AD有密切关系。在AD的相关因素（过敏性鼻炎、过敏性哮喘、经常皮肤瘙痒、经常出汗时瘙痒、全身皮肤干燥、身体屈侧皮炎、2岁前发病、初发于面部）中，基因型及等位基因频率均无显著性差别。基因型及等位基因频率在病人血清中的IgE水平高、低组间无显著性差异。3、AD是影响青少年儿童的常见病。2006年7～9月，两地总的患病率为0.98。男性标化患病率为1.19，女性标化患病率为0.82，差异无显著意义，P＞0.05。经年龄标化后沈阳市标化患病率为0.28％，其中沈阳地区男性标化患病率为0.52％，女性标化患病率，为0.05％，本溪地区标化患病率为1.08％，其中本溪地区男性标化患病率为1.24％，女性标化患病率为0.96％；沈阳本溪两地区间患病率比较没有显著性差异，χ²MH=1.325，P=0.250。经性别标化后，沈阳地区的患病率为0.16％，本溪地区的患病率为1.03％，两地患病率比较有显著的差异，χ²MH=6.977，P=0.008。AD是多因素共同作用导致，沈阳本溪地区AD的患病影响因素有年龄、性别、过敏性哮喘、过敏性鼻炎、全身皮肤干燥、经常皮肤瘙痒、经常出汗时皮肤瘙痒、家里潮湿、母亲分娩方式、家族异位症史等，其中经常过敏性哮喘、过敏性鼻炎、全身皮干、经常性皮肤瘙痒、经常皮肤出汗时瘙痒、家里潮湿、一级亲属有异位症是AD的危险因素，顺产是AD的保护性因素。更多还原

【Abstract】 Study on Epidermiology of Atopic Dermatitis and its association with Polymorphisms of SPINK5 and SCCE genes in Northern ChinaBackgroundAtopic dermatitis（AD） is a chronic inflammatory skin disease associated with cutaneous hyper-reactivity to environmental triggers. The clinical phenotype that characterizes atopic dermatitis is the product of interactions between susceptibility genes, the environment, defective skin barrier function, and immunologic responses. AD is often the first step in the atopic march that results in asthma and allergic rhinitis. Most of AD patients are adolescents and about 10% AD patients could come to their adulthood and there is tendency of regression along with the increase of age.It is necessary to get the message of the disease distribution, occurrence and development in population and race of people in order to study etiopathogenesis of AD.Atopic eczema affects more than one in ten children in developed countries, and the incidence is increasing. The prevalence of atopic eczema seems to be rising, but the factors responsible for this rise are not fully understood. Its prevalence has increased two- to three fold during the past three decades in industrialized countries but remains much lower in countries with predominantly rural or agricultural areas. Wide variations in prevalence have been observed within countries inhabited by groups with similar genetic backgrounds, suggesting that environmental factors play a critical role in determining expression of AD.The pathophysiology of eczema involves systemic as well as cutaneous immune and epidermal dysfunction. A precise understanding of the mechanisms underlying AD is critical for developmalet of more effective management strategies. Various studies indicate that AD has a complex etiology, with activation of multiple immunologic and inflammatory pathways。Atopic eczema is associated with epidermal barrier dysfunction. AD is characterized by dry skin and even involves nonlesional skin and increased transepidermal water loss. This impairmentt of the skin barrier function in AD leads to increased antigen absorption contributing to the cutaneous hyperreactivity characteristic of AD. The increased susceptibility to irritants in AD may therefore represent a primary defect of epidermal differentiation compounded by the presence of inflammation-induced skin damage.AD is a genetically complex disease that has a high familial occurrence. genetic factors play an important role in the development of this disease. Relevant to AD as part of a systemic atopic disorder, candidate genes involving the high affinity IgE receptor （FC RI-β）, the mast cell chymase gene, the cytokine gene cluster on chromosome 5q31-33, and the SPINK5 gene have been identified. Genome screens reported linkage of AD to loci on chromosomes 1q21, 17q25, and 20p.There has been particular focus on chromosome 5q31-33, as it contains a clustered family of Th2 cytokine genes, i.e., IL-3, IL-4, IL-5, IL-13, MGM-CSF and SPINK5. mutations in SPINK5 gene cause Netherton’s syndrome, a rare genodermatosis characteristised by an eczematous rash and raised IgE levels. Genome-wide screens in families with atopic eczema from frur different populations have detected several regions showing linkage with atopic eczema.Stratum corneum chymotryptic enzyme （SCCE） ,also named kallikrein 7, is a serine protease localized to the extracellular space of the stratum corneum and is specific for keratinizing cells undergoing desquamation. They are both highly expressed in granular keratinocytes and present in the intercellular spaces of the stratum corneum. Apart from its tissue localization, SCCE has several properties and characteristics, including its pH and inhibitor profile of catalytic activity, matching the basic prerequisites for a crucial involvement in desquamation under in vivo conditions. Transgenic mice overexpressing human SCCE developed changes in their skin similar to those seen in chronic atopic dermatitis.The overexpression of SCCE initially may lead to a premature breakdown of the epidermal barrier. The SCCE gene is therefore an important candidate gene for atopic dermatitis.In stratum corneum extracts,SCCE is responsible for a major part of the total proteolytic activity together with another serine protease, stratum corneum tryptic enzyme （kallikrein 5）.LEKTI is its inhibitors and proteolytic events is necessary for desquamation involving a tight balance between proteases and protease inhibitors in a particular microenvironment. SCCE may be up-regulated in chronic lesions of atopic dermatitis which could be associated with pathogenesis of inflamatory dermatitis which SCCE could be used as new target site for management.Subjects and Methods1 Association of SPINK5 gene with atopic dermatitis（1） Patients group:91 outpatients in No.1 Hospital of China Medical Uiversity were from around Shenyang. The diagnosis of AD was made according to Williams criteria. Serum IgE levels were determined by means of chemoluminescence.（2） Normal control group:250 normal controls who were volunteers of blood donor, were from Shenyang area .They denied a family history of AD,allergic rhinitis or asthma, and eczema, urticaria, etc.The blood samples were obtained from the Blood Transfusion service centre of Laoning province and stored at -80℃.Sex and age of subjects in the normal control group were well matched to the AD patients respectively.（3）MethodAnalysis of SPINK5 polymorphisms using polymerase chain reaction（PCR） and restriction fragment length polymorphism（RFLP） analysis. （1）. DNA Genome DNA was extracted from blood using phenyl-chloroform method.The sequence （Genebank NM₀06846）)of SPINK5 gene was retrieved from NCBI database.The fragments containing A1103G （Asn368Ser）, G1156A （Asp386Asn）, G1258A （ Glu420Lys ）, G2475T （Glu825Asp） were amplified by PCR using 10×buffer 2ul,0.5ul of forward primer and Reward primer, lul of genomic DNA,2ul of deoxyribonucleoside triphosphates（dNTP）, 1U of LA tap polymerase and deionized water was added to 20ul. The PCR conditions included initial denaturation for 3 min at 94℃,followed by 35 cycles at 94℃for 30s, 52℃～53℃for 30 min,72℃for 30s, followed by final extension at 72℃for 5 min. We genotyped this polymorphism by restriction fragmentt length polymorphism （RFLP） analysis. The products size is 367bp,367bp,356bp,325bp,respectively .The polymorphism were genotyped by restriction fragment length polymorphism（RFLP） analysis by restricted incision enzymes BtsⅠ,MboⅠ,HinfⅠ,FOKⅠrespectively. Digestion of PCR products were performed in 37℃waterbath for 10 hours and the products of amplied and restricted were run on a 2% agarose gel and analysed under UV light lamp.（2） A database was created and carried out using SPSS11.5 . Allele frequencies and genotypes were summarized .The genotype frequencies and allel frequencies were calculated respectively.Hardy-Weinberg equations were calculatde by the Chi-square test. Association of SPINK5 polymorphisms with AD was assessed using Chi-square test, single factor and Multiple logistic regression analyse. A 2-sided valre of p＜0.05 was regarded as statistically significance.2. Association of SCCE gene with atopic dermatitis（1）Patient group60 outpatients in No. 1 Hospital of China Medical Uiversity were from around Shenyang. The diagnosis of AD was made according to Williams criteria. Serum IgE levels were determined by means of chemoluminescence.（2）. Normal control group 120 normal controls who were volunteers of blood donor, were from Shenyang area .They denied a family history of AD,allergic rhinitis or asthma, and eczema, urticaria, etc.The blood samples were obtained from the Blood Transfusion service centre of Laoning province and stored at -80℃.Sex and age of subjects in the normal control group were well matched to the AD patients respectively.（3）Allele-specific PCR for genotyping.DNA Genome DNA was extracted from blood using phenyl-chloroform method.The sequence （Genebank NM₀06846）)of SCCE gene was retrieved from NCBI database. PCR-based assay was designed, using specific primers that discriminate between individuals that harbor the one-repeat （AACC） allele and those that harbor the two-repeat （AACCAACC） allele. The primers were designed such that they would be complemaletary to the DNA sequence, being exactly the same as each other but with the difference that one of them would miss the second repeat （AACC）.The fragments containing the allele were amplified by PCR using 10×Labuffer 2ul,0.5ul of forward primer and reward primer,1ul of genomic DNA,2ul of deoxyribonucleoside triphosphates（dNTP）, 1U of LA tap polymerase and deionized water was added to 20ul. The PCR conditions included initial denaturation for 3 min at 94℃,followed by 35 cycles at 94℃for 30s,58℃for 30 min,72℃for 30s,followed by final extension at 72℃for 5 min. The products amplied were run on a 2% agarose gel and analysed under UV light lamp.（4） statistical analysisA database was created and carried out using SPSS 11.5. Allele frequencies and genotypes were summarized .The genotype frequencies and allel frequencies are calculated respectively.Hardy-Weinberg equations were calculatde by the Chi-square test. Association of SCCe polymorphisms with AD was assessed using Chi-square test ,single factor and Multiple logistic regression analyses. A 2-sided value of p＜0.05 was regarded as statistically significance. 3、Epidemiology of AD in Northern China（1） subjectSubjects enrolled were students from No.85, 86, 90 middle school ,Hunan First Primary School, young kindergarden of Shenyang university ,Experimental Primary School of Benxi and The Second High School of Benxi respectively.（2） methodEpidemiologic survey was carryed out using questionnaire which is composed of 5 questions. AD were diagnosed with two or more of that on the basis of itch.. In addition,there were general state of health in questionary(age,gender, family medical history,inhabited environment ,treatment etc.There was concrete explanation for itch and flexor aspect in questionnaire.The questionnaires was handed out to the class by school doctors and claimed the students parents filling in questionnaire truly or assisting to those. Professional investigator （attending physician who has worked for years in dermatology clinic）examed the questionnairs and ample drawing and medical examination were carryed out.Results1. Association of SPINK5 gene with atopic dermatitis（1） We studied 91 patients with AD, aged 1-67 years（median 15±17）.The propotion of male aged ranging 1～10 years old was higher than that of female and the propotion of female aged ranging 31～67 years old was higher than that of male.There were 39 patients with higher level serum IgE than normal level’s and no significant difference was found among different groups.The distribution of male and female were balanced in those with related factors of AD such as allergic rithnitis,allergic asthma, skin pruritus,skin pruritus accompanying sweat, asteatosis cutis throughout body, flexor aspect dermatitis, AD onseting before 2 years old, AD onseting on cheeks. No significant difference was found in those with and without a history of family atopy. As controls,we studied 250 healthy subjects aged 19-73 years（median 27±8）,140 male ??and 110 female （1.27:1）. Distribution of male and female was balanced between patients and normal controls.（2）The allele frequency of SPINK5 in the patients were 0.63 for 1103G, 0.44 for 1156A, 0.54 for 1258A, 0.60 for 2475T, and Those in normal groups were 0.47 for 1103G, 0.45 for 1156A, 0.48 for 1258A, 0.47 for 2475T. There was significant difference in allele frequencies of SPINK5 2475T between the two groups, x²=4.697, P=0.03 and there were no significant difference in frequencies of 1103G, 1156A, 1258A among the two groups, P＞0.05. There were significant differences in genotype frequencies of SPINK5 1103A＞G, 1258G＞A, 2475G＞T between the two groups （P=0.000,0.000,0.035, respectively） and there was no significant difference in genotype frequencies of 1156G＞T （P＞0.05）. There were significant differences in genotype frequency of 2475G＞T between the patients with allergic asthma and the patient without allergic asthma, x²=11.111, P=0.004. Genotype GT of SPINK5 G2475T is a risk factor （OR=7.744, 95%C.I: 2.080～28.835）.2. Association of SCCE gene with atopic dermatitis（1）General informationWe studied 60 patients with AD, aged 1-64 years（median 14.5±14.5）, 31 was males, 29 was female. There were 26 patients with higher level serum IgE than normal level’s and no significant difference was found among ages groups. The distribution of male and female were balanced in those with related factors of AD such as allergic rithrfitis, allergic asthma, skin pruritus, skin pruritus accompanying sweat, asteatosis cutis throughout body, flexor aspect dermatitis, AD onseting before 2 years old, AD onseting on cheeks. No significant difference was found in those with and without a history of family atopy. As controls, we studied 120 healthy subjects aged 19-73 years old（median 27±8）, 71 male and 49 female （1.45:1）. Distribution of male and female was balanced between patients and normal controls.（2）Comparision between the patients and controls The genotype frequency in patients were 0.12 for AACC/AACC, 0.08 for AACC/AACCAAACC, 0.78 for AACCAACC/AACCAACC and those in normol controls were 0.279 for AACC/AACC, 0.287 for AACC/AACCAAACC, 0.435 for AACCAACC/AACCAACC. The allele frequencies in patients were 0.41 for AACC and those in normal controls were 0.59 for AACCAACC/AACCAACC. There were significant differences in distribution of alleles between both patiemts and control groups, P=0.000.There were significant differencies between two groups in allele frequences of SCCE gene inserted by AACC and genotype frequencies.3. Epidemiological study of atopic dermatitis（1） 7000 people were enrolled in the study, 6794 copies of questionnaire were accepted, 654 copies were excluded because of wrong filling and 61.40 copies were used for analysis. Most of the subjects were from 6～16 years old. Significant difference were observed in age distribution between Shenyang area and Benxi area. Gender distrbution was different too. 2661 subjects were male and 2482 were female （880 male and 907 female in Shenyang and 1781 male and 2482 female in Benxi）. In all subjects, 4919 were Han Nationality, 1221 were minority ethnic groups including Man nationality, Hui nationality, Korean nationality, Mongolia, etc.（2） Prevalence of ADPrvalence of AD was 0.98%, among the total, male prevalence rate was 53.3%, female was 46.7%. There was no significant difference between that of male and female. The age rang of AD was from 3 to 20, and the age median is 17±3 years. The age median of male is 17±5 years, while the female age median is 17±2 years.By standardization of age and sex, the standardized prevalence of Shenyang is 0.28% and that of Benxi city is 1.08%. There is a significant difference between the two city prevalence. x²=16.633, P=0.000. By standardization of age, the standardized prevalence of male in Shenyang is a little higer than that of female, x²=4.053, P=0.049. There is no significant difference between the standardized prevalence of male and female, x²=0.544, P=0.461. The standardized prevalence of Benxi is higher than the total prevalence of the two cities, but there is no significant difference exist, p＞0.05. The standardized prevalence of Shenyang is lower than the total prevalence of the two district. The x² value is 4.499, p=0.040. ccording to the age layer, two group is obtained. Group 1:3 years to 12 years. Group 2: 13 to 20 years. The two group show that the prevalence of 3-12 years old group is higher than that of 13-20 years group, though statistically no difference. The prevalence of 3-12 years old Benxi group is higher than that of 13-20 years Shenyang group, and there is no significant difference, p＞0.05.（3） Comparison among those with different AD correlated factorsWhen accompanied with dry skin, allergic rhinitis, allergic asthma, flexor aspect dermatitis, skin pruritus and, pruritus symptom in the time of sweating was more common. There were no significant differences in the related factors between those of males and females. The results of Single-Factor analysis to the AD related factors showed that there was no significant difference between those AD patients with and without the related factors. Allergic rhinitis, allergic asthma, flexor aspect dermatitis, skin pruritus and sweating-related itching, were risk factors of AD. According to the skin eruption of AD patients, attack rate decreases as related to hand, joint and trunk, neck, feet, flexor aspect of the extremities, face, extensor aspect of the extremities. The prevalence of the body parts has no significant difference between males and females.Among 60 Atopic Dermatitis cases, 16 have first degree relatives with atopic symptoms, 5 have second degree relatives with atopic symptoms, 3 have third degree relatives with atopic symptoms are. Among 60 AD cases, 34 cases have parents who smoke. Compared with non-AD group, the smoking rate is not significant different （P＞0.05）. Humid environment and pet-feeding in domestic environment are risk factors, maternal breast-feeding and cesarean delivery are protective factors.Conclusion 1. The SPINK5 gene A1103G （Asn368Ser）、G1258A （Glu420Lys）、G2475T （Glu825Asp） locus polymorphism has a close relationship with AD. The genotype and allele frequency have no significant difference between the population with or without the following correlated factors such as allergic rhinitis, allergic asthma, skin pruritus, sweating-related itching, dry skin, flexor aspect dermatitis, onset before 2 years old, initiating from face, etc. The GT genotype of gene locus G2475T is a risk factor for AD with allergic asthma2. SCCE gene 3’UTR domain AACC insert polymorphism has a close relationship with AD. The genotype and allele frequency have no significant difference between the population with the following correlated factors such as allergic rhinitis, allergic asthma, skin pruritus, sweating-related itching, dry skin, flexor aspect dermatitis, onset before 2 years old, initiating from face, etc. The genotype and allele frequency have no significant difference between the higher IgE AD group and the lower IgE AD group.3. AD is a common disease mainly affecting teenagers and children. The total standardized prevaled of the two cities in July 2007 is 0.98%, male standardized prevalenceis 1.19%, and the female standardized prevalence is 0.82%. There were no significantly differents, P＞0.05. AD is a result as of multiple factors. The influential factor of Shenyang and Benxi include age, sex, pet, humidity, breast-feeding, delivery pattern, family atopic history, etc. It has not been reported yet that delivery pattern has effect on AD.更多还原