中国生殖器疣和子宫颈癌HPV感染型别流行病学研究

【作者】 陈汶；

【导师】 李辉； 乔友林；

【作者基本信息】 北京协和医学院 ， 流行病与卫生统计， 2010， 博士

【摘要】 研究背景HPV (Human Papillomavirus人乳头状瘤病毒)病毒是危害人类健康的重要感染因素之一,它是引发生殖器疣和子宫颈癌的主要病因。2003年中国疾病预防控制中心发表数据显示,生殖器疣新发病例数为154,922,发病率为12.02/10万。世界卫生组织IARC 2004年发表的数据显示中国每年有45689例新发子宫颈浸润癌,占全世界子宫颈新发病例的十分之一。随着医学科技进展,已经研发出有效的针对HPV感染的基因重组疫苗(recombinant vaccine),目前疫苗主要针对的HPV型别包括HPV16、18、6和11型。由于还没有采用标准化的、高灵敏度检测方法获得全国HPV型别分布的流行病学数据,尚不能对该疫苗推广后可能产生的预防效果做出准确的评价。研究目的在中国不同地区开展以医院为基础的现况研究,调查具有生殖器疣、浸润性子宫颈癌(ICC)和CIN2以上的病例中HPV感染的流行情况和HPV的型别分布。材料与方法采用分层抽样方法选择研究对象,按照地理分布将全国分为东北、华北、西北、华中、西南、华南和华东7个大区,在每个大区遴选合作医院,共选择18家医院提供确诊生殖器疣病人及其标本和19家医院提供确诊子宫颈病变病人及其组织蜡块。疣体组织标本的HPV型别判定：采用酚-氯仿方法抽提DNA,使用通用引物MY09/11,MY09/GP5+和GP5+/GP6+以及巢式PCR扩增140-bp目标DNA。电泳分离后直接测序,如果是单一感染,直接用BLAST 2.0判定HPV型别；如果是多重感染,使用T-vector转化E.coli.,随机挑选五个克隆后测序,判定HPV型别。宫颈病变组织蜡块标本的HPV型别判定：用三明治切片法,第一张和最后一张蜡卷用于HE染色,中间蜡卷用于DNA提取。HE切片由病理专家组完成诊断。将经过病理确认合格的蜡卷与蛋白酶k溶液混合,70度过夜消化,以SPF10为引物扩增,用DNA酶免疫分析(DEIA)判定是否存在HPV感染,用SPF10 LiPA25 (version1)试剂盒对扩增产物进行型别判断。结果本研究获得1295例确诊的生殖器疣病人及其福尔马林固定的标本。因PCR扩增抑制或没有提取出足够量DNA,排除290例病人的标本,余下1005例病人标本符合本次实验的要求,用于本次的生殖器疣HPV感染的分析研究。生殖器疣患者的平均年龄为32岁,女性占55.3%(556/1005)多于男性的44.7%(449/1005)。1005例生殖器疣病人中HPV阳性者为891例,感染率为88.7%。在HPV阳性病例中,HPV6和HPV11分别占46.6%和42.4%,是最常见的HPV型别,其次是HPV16占11.8%；其它型别在HPV阳性病例中的比例均小于1%。891例病人中,单一型别HPV阳性率为97.2%,而多重感染比例仅为2.8%。多重感染的型别包括HPV6、11和16。HPV6、11和16型感染占全部生殖器疣HPV阳性病例的97.9%。未发现不同大区、城市和农村、不同性别和年龄组间HPV6和11型别分布存在统计学意义的差别。本次研究共获得664例ICC病人,其中鳞癌(SCC)630例、腺癌(ADC)34例,并获得CIN 2/3病人569例。SCC病人HPV感染率为97.6%,ADC为85.3%,CIN2/3为98.9%。HPV16(76.7%)和HPV18(7.8%)是SCC病人中最常见的感染型别,两者合计占SCC病人的84.0%；其次是HPV31占3.2%,HPV52占2.2%,HPV58占2.2%。在不同地区间SCC病人HPV感染率差别没有统计学显著意义。但是,SCC病人中,≤49岁年龄组的HPV16感染率大于50岁年龄组(P=0.003),而HPV52,58和39在高年龄组有更高的感染率；相反在CIN2和CIN3病人中,≤34岁和35-49岁年龄组HPV16感染率小于50岁年龄组(P=0.01),而HPV52、58和39在低年龄组感染率更高。与SCC病人相比,CIN2/3病人有更高的HPV31、52和58感染率,而HPV16和18的感染率则低SCC病人。结论HPV16和18是子宫颈病变病人中的最常见的型别,HPV6、11和16是生殖器疣病人中最常见的型别。提示目前的预防性疫苗能够达到很好的效果。更多还原

【Abstract】 Backgroud HPV (Human Papillomavirus) is an important infectious agent that is related to cervical precancerous lesion, cervical cancer and genital warts etiologically. Tha data from China CDC National Center for AIDS/STD showed 154,922 new CA patients in 2003 (12.02/100,000). The data published by IARC WHO in 2004 shows 45,689 new invasive cancers comes from China, accounting for one tenth in whole world. With the development of biomolecular technology, recombinant vaccines against HPV, which can prevent HPV infection effectively, are available in the market. There are no evidences from well-designed epidemiology study using standardized and sensitive technology for HPV detection, which can facilitates accurate evaluation on prophylactic vaccine’s effects.Objectives Hospital-based epidemiology study across China are carried out to study HPV distribution in genital warts, invasive cervical cancer (ICC) and CIN2/3 samples.Material and Methods Stratified sampling method was applied to get representive samples of the cases from the hospitals in 7 geographic regions, including Northeast, North, Northwest, Central, Southwest, South and East. Genital warts from the cases were provide by 18 hopitals, and archived paraffin blocks for cervical lesions are provided by 19 hospitals.We employed standard phenol-chloroform extracting and ethanol precipitation procedures on genital wards to refine DNA. Consensus primers MY09/11, MY09/GP5+ and GP5+/GP6+and nested PCR are applied to amplify 140 bp target DNA segment. For single infection, the amplimor is separated by electrophoresis and sequenced. For multiple infections, the amplimor is inserted into T-vector for E. coli. transformation. Five single clones were randomly selected for sequencing separately. Sequence information was blasted with NCBI database to ascertain HPV types.A sandwich technique was used to cut paraffin sections for Haematoxylin and Eosin (H&E) staining and paraffin sections for HPV DNA analysis from each cervical specimen. After confirmation of histological diagnosis, qualified cervical specimens were digested in Proteinase K buffer at 70 centigrade. SPF10 PCR on the 1:10 diluted DNA was applied to amplify HPV DNA and then the generic amplification products were detected by DNA enzyme immunoassay (DEIA). HPV-positive specimens were typed by reverse hybridization line probe assay SPF10 LiPA25 (version 1). Results Among 1295 genital warts patients with genital tissues collected,290 subjects were excluded due to no beta-globin and cyt B amplification, or PCR inhibition, or insufficient DNA for further testing and 1005 subjects were qualified for this study. The mean age is 32 years, female GW patients (55.3%; 556/1005) are more than male (44.7%; 449/1005). HPV prevalence was 88.7%(N=1005); HPV6 (46.6%) and HPV11 (42.4%) were the most common HPV types, accounting for 87.3%, followed by HPV16 (11.8%). Other HPV types were less than 1.0%. Besides, single infection was 97.2% of the total HPV positive GW patients, comparing with 2.8% multiple infections composed of HPV6, HPV11 and/or HPV16. Cumulative prevalence of HPV 6,11, and 16 is 97.9%. Neither HPV6 nor HPV11 significantly differed by urban and rural, sex, age levels and geographical regions.Archived paraffin blocks for 664 ICC (630 squamous cell carcinoma [SCC]; 34 adenocarcinoma [ADC]),569 CIN 2/3 cases from 7 regions of China were collected. HPV prevalence was 97.6% in SCC,85.3% in adenocarcinoma,98.9% in CIN 2/3. HPV16 (76.7%) and HPV18 (7.8%) were the most common, together accounting for 84.0% of SCC, followed by HPV31 (3.2%), HPV52 (2.2%), and HPV58 (2.2%). HPV prevalence in SCC did not differ notably by region. However, SCC cases from women≤34 years and 35-49 years had significantely higher HPV 16 prevalence than women over 50 years(P=0.003), among whom HPV52,58, and 39 were more common. In contrast to SCC, HPV16 prevalence in women over 50 years is singnificantely more than women≤34 years and 35-49 years, among whom HPV52,58, and 39 were more common. HPV16 and 18 were under-represented, whereas HPV31,52 and 58 were over-represented in CIN2/3(P=0.01),.Conclusion HPV 16 and 18 are the most common types for cervcia cancer, and HPV 6, 11 and 18 are the most common types for genital warts. It was suggested that the potential impact of prophylactic HPV vaccines is estimated to be high.更多还原