聚合酶链反应和斑点杂交方法检测人乳头瘤病毒DNA

目前用于检测ＨＰＶ感染的方法很多，我们对聚合酶链反应（ＰＣＲ）和斑点杂交方法进行了比较研究。一、临床资料１７例尖锐湿疣病例均来自１９９０～１９９１年我院皮肤科和妇科外阴门诊，５％醋酸白试验均阳性。所有病例均取病损活检，经病理诊断为尖锐湿疣。二、方法（一）ＰＣＲ１．寡核着酸引物的设计：根据已公布的病毒基因组序列设计寡核着酸引物序列，扩增４型ＨＰＶ的高保守区Ｅ６区［‘·’］。由于尖锐湿疣主要是ＨＰＶ６、１１型引起，ＨＰＶ６和１１型的基因组Ｅ６区有同源序列，故将ＨＰＶ６、１１型设计了一对共同引物，引物…     

聚合酶链反应检测尖锐湿疣皮损内人乳头瘤病毒

１３个皮损组织取自１３例尖锐湿疣（ＣＡ）患者，１３例基底细胞上皮瘤（ＢＣＣ）组织作为对照，用溴化钠液分离表真皮，从组织中提出的 ＤＮＡ和溴化钠液分别用聚合酶链反应检测 ＨＰＶ ＤＮＡ。１１例ＣＡ表皮中检出 ＨＰＶ ＤＮＡ，ＨＰＶ６有７例，ＨＰＶ１１有４例。３例ＣＡ真皮中发现有ＨＰＶ ＤＮＡ，ＨＰＶ６有２例，ＨＰＶ１１有１例。２例标本因溴化钠液被ＨＰＶ污染未作统计。在ＢＣＣ组织中未发现有ＨＰＶ ＤＮＡ。在某些ＣＡ真皮内含有 ＨＰＶ ＤＮＡ可以解释散发病例的复发性。     

人类乳头瘤病毒16全基因在体外培养的正常人角质形成细胞 …

目的 利用含有人类乳头瘤病毒（ＨＰＶ）１６全基因的质粒转染正常人角质形成细胞，观察ＨＰＶ１６ｍＲＮＡ在转染细胞的表达。方法 用ＦｕＧＥＮＥ＾ＴＭ６转染试剂，将携带ＨＰＶ１６全基因的质粒ｐＳＶ２－ｎｅｏ／１６转染体外培养的正常人角质形成细胞，在转染后２４ｈ提取细胞总ＲＮＡ和ＤＮＡ，进行ＲＴ－ＰＣＲ和Ｓｏｕｔｈｅｒｎ印迹分析，结果 ２４ｈ后，ＲＴ－ＰＣＲ成功地扩增出１１０ｂｐ的片段，转染细胞中已出现Ｈ     

原位杂交法检测尖锐湿疣中人乳头瘤病毒DNA型别

以非放射性地高辛配基标记人乳头瘤病毒（ＨＰＶ）６ｂ、１１、１６、１８型探针，用原位杂交法检测了重庆地区５０例经临床病理学诊断为尖锐湿疣的石蜡组织切片中ＨＰＶＤＮＡ型别，杂交条件分别采用严格条件（Ｔｍ－１２℃）和非严格条件（Ｔｍ－３５℃）进行。结果：尖锐湿疣中ＨＰＶＤＮＡ阳性率为８２％，其中ＨＰＶ６ｂ、１１、１６型分别为６２％、２４％、１０％，ＨＰＶ１８型未检出。杂交信号主要分布于表皮浅中层，且许多分布于空泡细胞内，但并非所有杂交信号均在空泡细胞内。     

2063例人乳头瘤病毒原位杂交病理形态学分析

目的 探讨ＨＰＶ－１／１１感染与组织病理形态改变之间的联系及其诊断价值。方法 应用原位杂交方法对２０６３例标本中ＨＰＶ－６／１１ＤＮＡ进行检测，并对凹空细胞进行流行病学评价。结果 ２０６３例中有１４６６例阳性，阳性率７１．０６％，尖锐湿疣组、增生组、乳头状瘤组和食上皮组的阳性率分别为９６．１６％、５９．８６％和６９．５７％。以凹空细胞作为诊断标准，其灵敏度为７７．９３％，特异度为９５．２６％，阳性     

人乳头瘤病毒及癌基因在人生殖器癌发生中的作用

我们对生殖器疣与癌中人乳头瘤病毒型别别分布及癌基因表达进行了研究。结果：（１）９８例生殖哭疣中ＨＰＶ６，１１的检出率为９５％，４３例生殖器癌中ＨＰＶ１６检出率为８８．３％；（２）Ｃ－Ｈａ－ｒａｓ及Ｃｍｙｓ癌基因在阴茎癌及宫颈癌中表达较生殖器疣中均显著增强（Ｐ＜０．００１）；（３）ＨＰＶ６感染病例ｒａｓ，ｍｙｃ基因表达率分别为１６．７％、６．７％，ＨＰＶ１１分别为１０％、２０％、ＨＰＶ１６分别为７０     

生殖器上皮HPV感染皮损中p53蛋白过度表达的检测

采用免疫组化方法对６例鲍温样丘疹病和９例尖锐湿疣生殖器上皮人类乳头瘤病毒（ＨＰＶ）感染皮损进行了ｐ５３蛋白、ｐ２１蛋白和增殖细胞核抗原（ＰＣＮＡ）表达的检测。结果ｐ５３阳性率为：鲍温样丘疹病（３／６）、尖锐湿疣（６／９），ｐ５３阳性细胞多位于基底层以及棘细胞层的中下层，同时发现受ＨＰＶ感染的空泡样变性细胞核中有ｐ５３蛋白和ＰＣＮＡ的过度表达，说明ｐ５３蛋白表达与ＨＰＶ感染及角朊细胞异常增生有关。     

尖锐湿疣组织中人乳头瘤病毒检测和白介素6表达

尖锐湿疣组织中人乳头瘤病毒检测和白介素６表达徐廷香李西启王剑波邹积才我们应用原位杂交技术，对尖锐湿疣（ＣＡ）组织中人乳头瘤病毒（ＨＰＶ）６．１１ＤＮＡ进行检测，并观察了ＨＰＶ分布特点及其与ＣＡ病理形态改变的关系，同时应用免疫组化方法对ＣＡ组织中白介素...     

含人类乳头瘤病毒16E7—L1的嵌合型病毒样颗粒的构建,表达和纯化 …

目的 研究人类乳头瘤病毒（ＨＰＶ）１６型的早期基因Ｅ７,构建、表达并纯化鉴定了６Ｅ７与ＢＰＶＬ１重组的嵌合型病毒样颗粒ＶＬＰＳ。方法ＨＰＶ１６Ｅ７基因分３段经ＰＣＲ增扩后分别克隆入连有ＢＰＶＬ１的质粒ＰＵＣ形成ＢＰＶＬ１－ＨＰＶ１６Ｅ７;以质粒ＰＶＬ１３９３为载体将ＢＰＶＬ１－ＨＰＶ１６Ｅ７基因转染杆状病毒并在昆虫细胞中进行表达;用超声粉和蔗糖超高、氯化工离心等方法纯化以及免疫印迹、ＥＣＬ、透射电     

尖锐湿疣新鲜组织HPV的聚合酶链反应检测与斑点杂交比较的研究

人乳头瘤病毒（ＨＰＶ）６／１１型是尖锐湿疣的主要病原体［１～３］。根据文献报导，以往检测方法很多，均无特异性，故从病原体的角度进行检测，具有重要的意义。我们将临床诊断力尖锐湿疣的患者７７例新鲜组织标本，用ＰＣＲ法检测ＨＰＶ６／１１型ＤＮＡ，并与斑点杂交法进行比较，现总结如下。一、病例选择ＣＡ患者共７７例，男４０例，女３７例，年龄１８～５６岁，平均３７岁，已婚４５例。病程最长者１年，最短１５天。发病部位：大小阴唇、阴道口、尿道口、龟头、冠状沟、包皮、肛ｌ’１周围等处。二、方法（一）模板ＤＮＡ提取：…     

ULTRASTRUCTURE AND HUMAN PAPILLOMAVIRUS DNA IN PAPILLOMATOSIS OF EXTERNAL AUDITORY CANAL

Background. A viral etiology has been suspected in papillomatosis of the external auditory canal (peac ), but virus particles have not been detected so far, although they are easily demonstrable in skin warts. The purpose of the study was to solve this discrepancy by the use of polymerase chain reaction (pcr ). Materials and Methods. Specimens from the external auditory canal of 14 patients with PEAC, but no human papilloma virus infection of the genital areas, were examined histologically by light and electron microscopy, as well as by PCR to detect viral dna . Results. Histologically, papillomatosis was present in all specimens. Vacuolated cells were found in the upper part of the stratum malpighii in five cases. On electron microscopy, the numbers of perichromatin and interchromatin granules were increased, but no viral granules were observed. In all specimens, dna of hpv 6 was detected using PCR, but there was no evidence of DNA of other hpv . Conclusions. Papilloma of the external auditory canal is produced by infection with hpv 6.     

性病门诊患者HPV-DNA荧光定量检测结果分析

目的分析性病门诊患者HPV-DNA荧光定量检测结果。方法采用荧光定量聚合酶链反应技术(FQ-PCR)对102例性病门诊患者进行HPV-DNA分型检测。结果 102例患者中HPV6/11型阳性率为27.66%,HPV16/18型阳性率为9.09%,分型结果以HPV6/11型为主,而HPV6/11型感染主要集中在20~40岁,其中20~30岁阳性率最高;HPV16/18型感染年龄均在40岁以上,其中以41~50岁较高,男、女HPV阳性率分别为7.89%和23.44%,差异有显著性。结论 HPV6/11型是HPV感染的主要型别,荧光定量聚合酶链反应(FQ-PCR)可作为尖锐湿疣诊断的指标,应加强对性病门诊患者的HPV检测。     

No Evidence of Human Papilloma Virus Infection in Basal Cell Carcinoma

Background:

Basal cell carcinoma (BCC) is the most common skin cancer among whites, and several risk factors have been discussed in itsdevelopment and progress. Detection of human papilloma virus (HPV) deoxyribonucleic acid (DNA) BCCs in some studies suggests that the virus may play a role in the pathogenesis of this disease. Several molecular studies showed conflicting reports.

Aims:

The purpose of this study was to investigate the association between HPV and BCC using polymerase chain reaction (PCR).

Materials and Methods:

HPV DNA detection was done for 42 paraffin-embedded tissue specimens of BCC and 42 normal skin samples around the lesions by PCR using GP5+/GP6+ primers.

Results:

HPV DNA was not found in any of the 42 samples of BCC, and only one normal skin sample around the lesions was positive for HPV DNA by PCR.

Conclusion:

In this study, no statistically significant difference was seen between the presence of HPV DNA in BCC and normal skin around the lesion, and HPV is not likely to have an important role in pathogenesis of BCC.     

尖锐湿疣人乳头瘤病毒颗粒、核酸及抗原检测

42例尖锐湿疣进行了HPV-DNA序列,HPV抗原和电镜检查。36例(85.7%)HPV-DNA序列检测阳性。其中单一型感染9例,混合型感染27例。以HPV_(11)阳性最多,计32例,HPV_(6b)26例,HPV_(16)19例,HPV_(18)6例。HPV抗原阳性28例(66.7%)。15例查见病毒颗拉(35.7%)。总计阳性率为90.5%(38/42例)。文中对三项检查结果作了比较和分析。     

Detection of human papillomavirus in nongenital Bowen's disease by in situ DNA hybridization

Genital Bowen's disease has been strongly linked in recent studies to human papillomavirus (HPV). Nongenital Bowen's disease has been less well investigated, although isolated reports, all of which involved detection of HPV after extraction of DNA from fresh-frozen tissue, have been made. We investigated 25 cases of nongenital Bowen's disease in 5 black and 20 white patients for the presence of HPV types 1, 6, 11, 16, and 18 using paraffin-embedded tissues. Human papillomavirus was present in six specimens from 3 of the 5 black patients (one previously reported to be positive on Southern blot) and 3 of the 20 white patients; HPV 16 was detected in all 6 cases on low-stringency testing, but only 4 remained positive on high-stringency testing. This suggests an HPV closely related to but not entirely homologous with HPV 16 in the 2 remaining cases. Five of the 6 positive specimens were lesions from the hands and feet and 1 was from the volar aspect of the arm. Clinical factors associated with the presence of HPV included black race, location on the palmar surface and the feet, young age, and verrucous or hyperkeratotic clinical appearance. Of the 6 positive cases, all 5 of the patients available for examination also had evidence of HPV-associated genital lesions. No specific histopathologic features were found to be indicative of the presence or absence of HPV.     

Human papillomavirus DNA detection in primary anogenital warts and cervical low-grade intraepithelial neoplasias in adults by in situ hybridization.

In this study, 58 consecutive patients with primary anogenital warts were selected from patients attending a genitourinary clinic. Patients were grouped on the basis of clinical lesion site, i.e. patients with genital warts only, patients with perianal or anal canal warts only, and patients with concurrent perianal and genital warts. Of these patients, 38% of the men (12/31) and 33.3% of the women (9/27) had other anogenital infections, such as nonspecific urethritis (NSU) or nonspecific genital infection, which were the most common. Of the patients who had perianal warts, 37% of the men (7/19) and 25% of the women (4/16) also had warts in the anal canal. Of the women who had anogenital warts, 63% (17/27) had concurrent subclinical low-grade cervical intraepithelial neoplasia (CIN) lesions. Human papilloma virus (HPV) DNA (either 6 or 11, 16 or 18, or 31 or 33 or 35) was detected in 53.3% (40/75) of the anogenital wart biopsy samples, and in 35.2% (6/17) of the low-grade CIN lesions. HPV types 6 or 11 were the most common types in anogenital warts (45.3%); and in CIN lesions HPV types 6 or 11 and 16 or 18 were found with equal frequency (17.6% each). There were no significant differences in HPV types between patients with genital warts and patients with perianal and anal canal warts. Anogenital infection with HPV is multicentric; external anogenital warts and subclinical CIN lesions often exist concurrently. The low prevalence of HPV DNA detected in anogenital warts and CIN biopsy samples may be due to insensitivity of the in situ hybridization technique used in this study.     

A new type of human papillomavirus associated with oral focal epithelial hyperplasia

Lesions from 10 patients suffering from focal epithelial hyperplasia (FEH) of the oral mucosa, including those of 4 Greenlandic Eskimos, were investigated for the presence of human papillomavirus (HPV) DNA sequences by blot hybridization experiments. Two distinct HPVs were detected in the DNA extracted from these lesions, and their genomes were molecularly cloned and characterized. One of these HPVs, detected in 4 patients, was found to be identical with HPV13, whose association with FEH was already known. The other one, detected in 6 patients, was only weakly related to HPV13 and to the other HPVs associated with lesions of the mucous membranes, and constituted a new HPV type, tentatively named HPV32. Lesions from other types of oral papillomas, obtained from 14 additional patients, were also analyzed. Human papillomavirus DNA sequences were detected in the DNA preparations extracted from 5 specimens: HPV6 DNA in a condyloma and in a papilloma, 2 as yet uncharacterized HPV DNAs in 2 papillomas, and HPV32 DNA in a papilloma which showed histologic similarities to FEH. Thus, it seems likely that FEH of the oral mucosa is a disease associated with 2 specific HPVs--HPV13 and HPV32.     

In situ DNA hybridization analysis of human papillomavirus (HPV) sequences in benign oral mucosal lesions

Summary A series of 144 surgically treated benign oral mucosal lesions were analysed using an in situ DNA hybridization technique with ³⁵S-labeled human papillomavirus (HPV) DNA probes to demonstrate the DNA of HPV types 6, 11, 13, and 16, in routinely processed, paraffin-embedded biopsy specimens. These lesions and an additional 62 benign oral mucosal biopsy specimens (total, 206 specimens) were also assessed by the indirect immunoperoxidase (IP-PAP) technique to detect the expression of HPV structural proteins (viral antigens). A total of 54/206 (26.2%) lesions were observed to express HPV antigens, being found in 45/92 (48.9%) of the squamous cell papillomas/condylomas, in 1/54 fibrous hyperplasias, in 1/8 true fibromas, and in 7/8 (87.5%) of the focal epithelial hyperplasia (FEH) lesions. Of the HPV DNA-positive lesions, 15/45 (33.3%) expressed HPV antigens, the expression not being related to any particular HPV type. HPV DNA sequences were found in 45/144 (31.3%) of the lesions. HPV DNA was present with the highest frequency in FEH (83.3%), papillary hyperplasia (28.6%), fibrous hyperplasia (24.4%), and true fibromas (14.3%). The most frequent HPV type was HPV 11, representing 37.8% of the DNA-positive lesions. HPV 13 DNA, previously regarded as specific to FEH, was disclosed as a single HPV type in seven cases, and as a double infection by HPV 11 and 13 in an additional three cases, including all five morphologically distinct entities. Noteworthy is the discovery, of the high-risk HPV type 16 DNA in 17.8% of the DNA-positive lesions, four papilloma/condyloma lesions, three fibrous hyperplasias, and one FEH. The results confirm the previously reported evidence regarding HPV involvement in oral mucosal lesions. The implications of these findings are discussed in terms of the epidemiology, HPV etiology, and proper classification of the oral mucosal lesions, with special emphasis on the discovery of the high-risk HPV type 16 in the benign lesions as well as in oral cancer. The use of the in situ DNA hybridization as a powerful tool in detecting the specific HPV DNA sequences in routinely processed oral biopsy specimens is strongly recommended.     

HPV-related cancer susceptibility and p53 codon 72 polymorphism

Conflicting results regarding the association of a polymorphism at codon 72 of the p53 tumour suppressor gene and susceptibility to develop human papilloma virus (HPV)-associated cervical cancer have been published over the last year, implicating differences in ethnic background, sample origin, sample size and/or detection assay. The material for this study was collected in the identical geographical region as for 2 previous reports with contradictory results regarding the association of codon 72 genotype with squamous cell cancer (SCC). We have used an alternative detection assay, based on pyrosequencing technology, that interrogates the variable position by the accuracy of DNA polymerase. In addition to cervical clinical specimens from SCC, HPV16- and HPV18-infected adenocarcinoma cases as well as cervical intraepithelial neoplasia (CIN) were investigated. No significant association was found between p53 codon 72 genotype and the risk to develop adenocarcinoma, SCC or CIN in the Swedish population.