【作者】 孙彩虹；

【导师】 顾恒； 常宝珠； 李新宇； 陈崑；

【作者基本信息】 中国协和医科大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 疣是人乳头瘤病毒(HPV)感染所致常见的皮肤科疾病之一,在临床工作中,常遇到如下困惑:发生在指(趾)间、腋下、腹股沟或口腔内形态酷似尖锐湿疣的皮疹如何诊断?发生在肛门、外生殖器的类似寻常疣且数年无变化的皮疹是否也诊断为尖锐湿疣?其HPV型别与寻常疣相同还是与尖锐湿疣相同?非生殖器部位皮损是否存在高危型HPV感染的可能?继发肿瘤的机会有多大?本研究拟通过了解此类“特殊皮肤疣”的形态、病理、HPV型别之间的相互关系,旨在回答临床工作中遇到的问题,寻找其分子生物学、临床治疗学和流行病学资料,为这组“特殊皮肤疣”诊治提供相应的理论基础,为预后提供一定的指导作用,并为进一步的深入研究奠定基础。全文具体分为三部分。第一部分“特殊皮肤疣”中人乳头瘤病毒的基因分型研究目的探讨特殊部位(趾间,腹股沟)皮肤疣、特殊形态(鸡冠状、菜花状)头面部疣和特殊形态(乳头状、丘疹状)生殖器疣等四组特殊皮肤疣的HPV基因型别分布。方法通过直接测序法对“特殊皮肤疣”中的HPV进行基因分型研究。根据测序所得到的型别,结合文献中皮肤疣的常见型别,分别设计型特异引物,通过型特异性PCR来验证和补充测序结果。结果(1)趾间组20例、腹股沟组29例,HPV型别以HPV7为主;头面部组30例HPV型别以HPV27、7为主;生殖器组18例HPV型别以HPV6、11为主,未检测到HPV7;HPV2、7、27、57是寻常疣组中HPV主要的型别,腹股沟组和生殖器组均出现高危型(HPV16)。与寻常疣对照组相比,趾间组、腹股沟组和生殖器组HPV7构成比差异有显著统计学意义(P值分别为0.000、0.000和0.011);而头面部组HPV7构成比差异无统计学意义(P=0.413)。(2)7对TSP-PCR的结果表明,在趾间、腹股沟、生殖器组均存在多种型别的混合感染。两种方法的型别符合率较高(80～99%)。结论趾间、腹股沟等皮肤皱褶处疣中HPV7的感染性较高,皮疹形态以菜花状、丘疹状为主。头面部菜花状疣HPV型别以HPV27、7为主,生殖器丘疹状疣HPV型别以HPV6、11为主,腹股沟疣和生殖器丘疹状疣中存在高危型HPV(HPV16)。各组均存在HPV混合型别感染。第二部分特殊皮肤疣的临床病理特征研究目的探讨“特殊皮肤疣”的临床和病理特征及各观察组的HPV基因型别与主要的临床病理特征之间的相关性。方法对趾间疣、腹股沟疣、头面部疣和生殖器疣四组“特殊”皮肤疣的临床和病理特征进行归纳和总结,对各观察组的HPV基因型别与主要的临床病理特征之间的相关性进行统计学分析。结果趾间组、腹股沟组患者均为男性,形态以菜花状、丘疹状为主,与寻常疣组形态相比,差异有统计学意义(菜花状P=0.011,丘疹状p=0.005)。疣体形态与HPV型别之间没有显著的关联性。共有56例患者的皮损作普通病理,表现寻常疣模式者18例,尖锐湿疣模式者16例,不易鉴别者22例。HPV7阳性(18份)的病理象特征:(1)乳头瘤样增生和棘层肥厚;(2)内含中等大小的透明角质颗粒的细胞深染;(3)显著的角化过度和角化不全。结论HPV型别与疣体形态之间的相关性无显著的统计学意义。特殊皮肤疣病理可表现为典型的寻常疣病理像或尖锐湿疣病理像,HPV7阳性的病理像具有特征性组织学改变。结合特殊皮肤疣中HPV型别分布,建议特殊部位(趾间,腹股沟)皮肤疣、特殊形态(鸡冠状、菜花状)头面部疣诊断为寻常疣;特殊形态(乳头状、丘疹状)生殖器疣诊断为尖锐湿疣。第三部分PCR产物的TA克隆及趾间皮屑HPV DNA的检测目的对趾间疣组中的基因型进行进一步验证,初步探索HPV7在趾间疣中的寄居或致病状态。方法采用PCR产物的TA克隆法对趾间疣组中的基因型进行进一步验证,设计趾间疣的病变阴性对照,取足癣患者和健康人群的趾间皮屑筛查有无HPV及HPV7的寄居。结果取趾间疣组12份DNA标本,TA克隆其通用引物PCR产物,每一份标本的4个TA克隆子中至少有1个成功合成质粒重组体,其测序所得序列与直接测序法所得的序列序均有90%以上的一致性。共采集趾间糜烂型足癣患者皮损处皮屑53例作为阴性对照和健康人群趾间皮屑48例作为空白对照,HPV的阳性率分别为(15.09%,8/53;16.67%,8/48),未检测到HPV7 DNA。结论测序型别可能是趾间疣的致病的HPV型别,趾间疣中所检测到的HPV7,可能处于致病状态,HPV7可感染免疫力正常的人群。更多还原

【Abstract】 In dermatology clinical work,we encountered patients with warts in between toes,in armpits,on the groin,cauliflower-like warts on head and face;papule-like warts in genital site.These cauliflower-like lesions were similar to condylomata acuminata and papule-like lesions were similar to verruca vulgaris in morphology.What was the exact diagnosis with these special warts? Was there any high-risk genotypes in these warts?—We have studies on these special warts to expore basic molecular epidemiological and the clinical and histological characteristics to help dermatologist answer the stumbles in clinic.The paper consists of three parts.Part 1 The distribution of the HPV genotype of human papillomavirus(HPV) infections among patients within special warts.Objectives To investigate the molecular epidemiological characteristics of human papillomavirus(HPV) infections among patients with special warts (warts in webspace or groin;cauliflower-like warts on head and face; papule-like warts in genital site),analyze the distribution of the HPV genotype.Methods DNA isolated from warts specimens were amplified by PCR and sequencing to identify HPV genotype.Type-special primer PCR(TSP-PCR) of HPV2,4,6,7,11,16,and 18 were also performed to compare the conformity of the two methods.Results(1) Ninety-five cases were enrolled in the study groups.The main genotype was HPV7 in interdigital group(20 cases) and groin group(29 cases).In head and face group(30 cases),HPV7 and HPV27 were the main genotype.HPV6 and HPV11 were the main genotypes in genital group(18 cases). HPV2,7,27,57 were the main genotypes in verruca vulgaris group(31 cases). And a high-risk genotype(HPV16) was detected in genital and groin group. There was significant difference between some groups and the verruca vulgaris control group in constituent ratio of HPV7(interdigital group P=0.000,groin group P=0.000,genital group P=0.011).There was no significant difference between head and face group and verruca vulgaris group in constituent ratio of HPV 7(P=0.413).(2)The results of 7 pairs TSP-PCR showed that a variety of mixed-genotypes infection existed in all groups.The conincidence rates were 80-99%in sequencing and TSP-PCR.Conclusions Warts located in webspace and groins are prone to be caused by HPV7.HPV7 and HPV27 are the main genotype in cauliflower-like warts on head and face.HPV6 and HPV11 the main genotypes in papule-like in genital warts.High risk HPV(HPV16) genetype DNA exists in genital and groin warts.Part 2 Clinical and histological features and the potential relationship between HPV genotypes and clinical characteristics in patients with these special warts.Objectives To expore the clinical and histological features and the potential relationship between HPV genotypes and clinical characteristics in patients with these special warts.Methods The clinical and histological characteristics of those warts,the relationship between HPV genotypes and its clinical and histological characteristics were analyzed and statisticsed by SPSS.Results The patients were all males,warts were mainly cauliflower-like and papule-like in interdigital group and groin group.There was significant difference between these two groups and the verruca vulgaris control group in gentile(P=0.000) and morphous(cauliflower-like,P=0.011;papule-like P=0.005).But there was no significant relationship between the morphology of the warts and HPV genotype.Fifty-six specimens were taken for analysis of histopathological patterns.It revealed 18 cases showing verruca vulgaris-like pathology patterns,16 condyloma accuminatum-like pathology features,and 22 cases could not determine verruca vulgaris-like or condyloma accuminatum-like pathology patterns.Main HPV7(9/18) positive samples’ pathology manifestations matched the characteristics for HPV7 infection,which showed papillomatosis and acanthosis,deep-stained cells including keratohyalin granules in middle size,and obvious hyperkeratosis and parakertosis.Conclusions Warts located in webspace and groins,mainly cauliflower-like and papule-like,are prone to offend male patients.But there is no significant relationship between the morphology and HPV type of the warts. Special warts could show verruca vulgaris-like or condyloma accuminatum-like pathology patterns.Part 3 TA clone of PCR products and detection of the HPV DNA in scales of webspace.Objectives To explore the role of HPV7 in the pathopoiesis of warts. Methods the gene sequence of HPV found in webspace specimens was studied with PCR and TA clone.The HPV and HPV7 DNA in scales of webspace in patients with tinea pedis and healthy persons were both detected by nest-PCR and TSP-PCR.Results The purified and amplified DNA of 12 specimens in interdigital group were cloned with the pUC57-T Vector Cloning Kit.Four recombinant plasmids harboring inserts of the expected size were randomly selected and sequenced. The result showed the sequence shared 90%or more homology to the HPV genotype in direct sequencing.HPV DNA was detected in scales specimen of the webspace in 53 patients with tinea pedis and 48 healthy persons.HPV DNA positivity rate was 5/53 and 5/48 respectively in nest PCR.There was no HPV7 positive in all scale specimens in TSP-PCR.The results revealed statistically significant difference in sex ratio(P=0.002),and the positivity rate of HPV(P=0.000) or HPV7(P=0.000) between interdigital and tinea pedis groups or between interdigital and healthy person groups.There was no significant difference between the tinea pedis group and healthy persons group in sex ratio(P=0.835) or positivity rate in HPV7(P=0.829).Conclusions HPV7 maybe plays a pathogenic role in the development of interdigital warts.It could induce warts in immunocompetent patients.更多还原