PCR和PCR产物斑点杂交技术检测成人咽喉病变中人乳头状瘤病毒感染

目的 :研究成人咽喉部良、恶性病变与人乳头状瘤病毒 (HPV)感染的关系。方法 :应用聚合酶链反应 (PCR)和斑点杂交技术 ,对 5 5例咽喉不同病变的新鲜组织标本进行 HPV6,1 1 ,1 6,1 8,33共 5型HPV- DNA感染的检测。结果 :在咽乳头状瘤组 HPV感染率为 6 0 % (6 / 1 0 ) ,喉乳头状瘤组为 70 % (7/1 0 ) ,喉鳞状上皮非典型增生组为 2 0 % (1 / 5 ) ,声带息肉组为 2 0 % (1 / 5 ) ,喉癌组为 2 0 % (1 / 5 ) ,声带小结组为 0 (0 / 1 0 )。HPV- DNA型别分布在咽喉良性病变中以 HPV6,1 1 型为主 ,喉癌中以 HPV1 6为主。结论 :本地区成人咽喉良、恶性病变的发生与 HPV感染密切相关。PCR结合核酸斑点杂交法检测 HPV,具有灵敏性高、特异性强的优点 ,值得推广应用。     

山东东部57例喉癌人乳头状瘤病毒DNA及其亚型的检测

目的 通过检测山东东部地区喉鳞状细胞癌中人乳头状瘤病毒(HPV)的DNA及其亚型的表达,探讨HPV感染与喉癌的关系及其在喉癌发病中的意义。方法 采用聚合酶链式反应(PCR)扩增和DNA反向点杂交相结合的DNA芯片技术,检测57例喉鳞状细胞瘤各亚型HPV DNA,对照组为同期喉乳头状瘤细胞8例。结果 喉癌组HPV感染率为7.02%(4/57),喉乳头状瘤组75.00%(6/8),差异有统计学意义(χ2=24.906,P＜0.01);喉癌组高危型HPV16与低危型HPV43感染率分别与喉乳头状瘤比较,差异均无统计学意义(χ2= 5.611、0.143,P＞0.05);喉乳头状瘤组低危型HPV11与HPV6感染率较喉癌组高,差异均有统计学意义(χ2=8.611、14.702,P＜0.05)。结论 山东东部地区喉癌患者HPV感染率较低,而低危型HPV6/11与喉乳头状瘤关系较为密切     

儿童喉乳头状瘤细胞的原代培养及生物学特性研究

目的 探讨人乳头状瘤病毒（human papilloma virus,HPV)感染阳性患儿喉乳头状瘤细胞在体外培养的生物学特性。方法 2000年3月－2001年4月采用组织块培养法培养10例12例次喉乳头状瘤患儿手术的标本－HPV感染阳性患儿喉乳头状瘤细胞，观察其生长情况，计数法绘制细胞生长曲线，运用共同引物聚合酶链反应（polymerase chain reaction,PCR)法及核酸分子斑点杂交方法对喉乳头状瘤细胞在培养前后的HPV DNA进行检测。结果 HPV感染阳性的喉乳头状瘤细胞在体外生长时间可长达6周，培养前后细胞内均含有HPV的DNA。细胞生长分为3期，延缓期、生长期及停滞期。培养前1－4d细胞从组织块中大量游出，5－7d为延缓期，此期间细胞渐渐开始贴壁，8－18d为生长期，细胞数目迅速增多，生长速度很快，继而进入停滞期，细胞数目增长缓慢，细胞空泡化明显，渐渐走向死亡。结论 HPV感染阳性的喉乳头状瘤细胞在体外生长情况良好，但要建立HPV感染阳性的喉乳头状瘤细胞的动物模型尚需进一步研究。     

喉乳头状瘤HPV16/18感染与p53蛋白表达的相关性

目的：了解喉乳头状瘤组织内ＨＰＶ１６／１８的感染与抑癌基因ｐ５３变异的关系,以及ＨＰＶ感染在喉乳头状瘤发病中的作用。方法：采用ＰＣＲ和免疫组化技术,检测３５例喉乳头状瘤组织中ＨＰＶ１６／１８ＤＮＡ及ｐ５３蛋白的表达。结果：２４例组织中检出ＨＰＶ１６／１８ＤＮＡ（６８．６％）;１９例ｐ５３蛋白呈过度表达（５４．３％）;在１２例中同时检出ＨＰＶ１６／１８ＤＮＡ和ｐ５３蛋白过度表达（３４．３％）。结论：     

儿童喉乳头状瘤细胞的原代培养及生物学特性研究

目的　探讨人乳头状瘤病毒 (humanpapillomavirus,HPV)感染阳性患儿喉乳头状瘤细胞在体外培养的生物学特性。方法　 2 0 0 0年 3月～ 2 0 0 1年 4月采用组织块培养法培养 10例 12例次喉乳头状瘤患儿手术的标本———HPV感染阳性患儿喉乳头状瘤细胞 ,观察其生长情况 ,计数法绘制细胞生长曲线 ,运用共同引物聚合酶链反应 (polymerasechainreaction ,PCR)法及核酸分子斑点杂交方法对喉乳头状瘤细胞在培养前后的HPVDNA进行检测。结果　HPV感染阳性的喉乳头状瘤细胞在体外生长时间可长达 6周 ,培养前后细胞内均含有HPV的DNA。细胞生长分为 3期 ,延缓期、生长期及停滞期。培养前 1～ 4d细胞从组织块中大量游出 ,5～ 7d为延缓期 ,此期间细胞渐渐开始贴壁 ,8～ 18d为生长期 ,细胞数目迅速增多 ,生长速度很快 ,继而进入停滞期 ,细胞数目增长缓慢 ,细胞空泡化明显 ,渐渐走向死亡。结论　HPV感染阳性的喉乳头状瘤细胞在体外生长情况良好 ,但要建立HPV感染阳性的喉乳头状瘤细胞的动物模型尚需进一步研究     

中耳癌人类乳头状瘤病毒DNA表达的研究

目的 :研究原发性中耳癌人类乳头状瘤病毒 (H PV) DNA的表达情况。方法 :采用多聚酶链反应技术 ,对 5例中耳癌的病理组织蜡块进行 HPV - DNA类型的检测 ,以 8例慢性化脓性中耳炎中耳乳突粘膜为对照组。结果 :5例中耳癌高危型 HPV感染率为 80 % (4 /5 ) ,以 HPV1 6型感染为主 ,而对照组 HPV感染率为 0 % (0 /8) ,经 χ2检验 ,P <0 .0 1。结论 :中耳癌高危型 HPV感染率较高 ,慢性化脓性中耳炎和中耳胆脂瘤以及 HPV感染可能在中耳癌的发生发展中起着重要作用     

人乳头状瘤病毒感染与鼻内翻性乳头状瘤的发病及恶变关系的研究

目的:探讨人乳头状瘤病毒(HPV)感染与鼻内翻性乳头状瘤(NIP)的发病及恶变的关系。方法:将57例NIP患者分为不伴恶变组(45例)和伴恶变组(12例),应用PCR技术,检测其HPV6、11、16、18等4个型别的HPV-DNA,同时以30例鼻息肉患者为对照组。结果:57例NIP患者HPV-DNA总阳性率为64.9%(37/57)。不伴恶变组HPV-DNA阳性率为60%(27/45),均为单一的低危型HPV11型感染;伴恶变组HPV-DNA阳性率为83.3%(10/12),以检出HPV16、18型DNA为主,其中5例为双重感染(4例为HPV16、18型,1例为HPV11、16型),4例为单一型HPV16型感染,1例为单一型HPV11型感染。而30例鼻息肉患者均未检出HPV-DNA。结论:HPV在NIP的发病中有重要作用,而高危型HPV16、18型与NIP恶变可能密切相关。     

婴幼儿咽喉乳头瘤组织人乳头瘤病毒感染的探讨

目的　探讨温州地区人乳头瘤病毒感染和婴幼儿咽喉乳头状瘤的关系。方法　应用聚合酶链反应和核酸斑点杂交技术检测 35例婴幼儿咽喉乳头瘤组织和 10例对照组组织 (小儿声带小结 )HPV6、11、16、18、3 3 5个型别的DNA。结果　乳头瘤组织HPV感染率为 91 4% (30 / 35 ) ,其中HPV6型检出率为 5 4 2 % (19/ 35 ) ,HPV11型感染率为 2 5 7% (9/ 35 ) ,多重型别HPV6 11感染率为 11 4% (4/ 35 ) ;HPV16、18、3 3 型 ,均为阴性。对照组各型检测结果均为阴性 ,两者对比差异有高度显著性 (P <0 0 0 1)。结论　温州地区婴幼儿咽喉乳头瘤的发生与HPV感染密切相关 ,尤以HPV6感染为主。PCR结合核酸斑点杂交技术检测HPV具有敏感性高、特异性强的优点 ,值得推广。     

不同类型鼻内乳头状瘤与HPV感染的关系

目的探讨不同类型的鼻内乳头状瘤与人乳头状瘤病毒(HPV)感染的关系.方法按照1991年WHO的上呼吸道肿瘤分类法,鼻内乳头状瘤病理标本30例中,外生性乳头状瘤7例,内翻性乳头状瘤18例,柱状细胞乳头状瘤5例.采用多聚酶链反应(PCR)和免疫组化(IH)法分别检测标本中与HPV相关的DNA和PV结构的抗原,并以5例鼻息肉为对照.结果7例外生性乳头状瘤组织中5例(72%)HPV阳性;18例内翻性乳头状瘤仅1例(5.8%)阳性;5例柱状细胞乳头状瘤及对照组鼻息肉5例中均未检出阳性.外生性乳头状瘤组中HPV的检出率与其他各组差异有显著性(P＜0.05).结论HPV感染与外生性乳头状瘤的发生过程密切相关,与内翻性乳头状瘤和柱状细胞乳头状瘤的病因无明显相关性.对内翻性乳头状瘤的治疗目前采用干扰素及转移因子等药物治疗效果有待进一步探讨,以手术治疗为主较妥.     

喉鳞状细胞癌和喉乳头状瘤中Survivin表达的意义

目的：探讨Survivin蛋白在喉鳞状细胞癌和成人喉乳头状瘤中的表达及其意义。方法：应用免疫组化方法对46例喉鳞状细胞癌、24例癌旁组织和20例喉乳头状瘤以及16例正常喉粘膜标本中Survivin的表达进行检测。结果：Survivin蛋白在喉癌组织、癌旁组织和喉乳头状瘤中的阳性表达率分别为71.74%（33/46）、33.33%（8/24）和40.0%（8/20）,在正常黏膜组织中没有表达,喉癌组中的阳性表达率分别显著高于正常组织、癌旁组织及喉乳头状瘤中的表达率（均P〈0.01）。且发生恶变的喉乳头状瘤Survivin蛋白均为阳性表达。但Sur-vivin阳性表达与喉癌的发生部位、分级、分期、淋巴结转移以及病理分级没有关联。结论：Survivin在喉癌组织中过度表达,参与了喉癌的形成,是喉癌形成的一个早期事件,可望成为预测成人喉乳头状瘤恶变的一个诊断方法。     

喉癌人乳头瘤病毒感染和p53蛋白表达的研究

对４４例喉鳞癌和１６例声带息肉患者采用原位核酸杂交技术探测其人乳头瘤病毒（ＨＰＶ）６Ｂ，１１，１６，１８型ＤＮＡ同源序列及ＬＳＡＢ免疫组化法探测其Ｐ５３蛋白的表达，结果：（１）喉癌与ＨＰＶ１６／１８杂交阳性率为４３．２％（１９／４４），声带息肉为１２．５％（２／１６）（Ｐ〈０．０５）（２）喉癌ｐ５３蛋白阳性率为５６．８％（２５／４４），声带息肉全部为阴性；（３）喉癌ＨＰＶ１６／１８杂交及ｐ５３蛋白     

Human papilloma virus and p53 expression in carcinomas associated with sinonasal papillomas: a Danish Epidemiological study 1980-1998

OBJECTIVES: To determine a putative role and relation between human papilloma virus (HPV) and p53 in the etiology of sinonasal carcinomas associated with papillomas. STUDY DESIGN: The study group consists of all patients with sinonasal carcinomas associated with papillomas diagnosed in Denmark from 1980 to 1998. After reviewing our national pathological files, tumor tissues from 36 patients were collected, comprising 15% of the total cases of sinonasal carcinomas. In 35 cases a squamous cell carcinoma was demonstrated and in one case an adenocarcinoma was evident. Inverted papilloma was associated with carcinoma in 31 cases and exophytic papillomas in 5 cases. The material was investigated for HPV using polymerase chain reaction analyses with two sets of consensus primers (GP5+/GP6+ and MY09/MY11). The HPV-positive cases were submitted to dot-blot hybridization to establish the HPV type. Using immunohistochemistry, the p53 expression was determined. A p53 overexpression is defined as positive staining in 10% or more of the tumor cells. RESULTS: Among 30 examined cases of carcinomas associated with inverted papillomas, 4 cases were HPV-positive (13%). P53 overexpression was not shown among the HPV-positive cases, whereas p53 overexpression was seen in 21 of the 24 (88%) examined HPV-negative cases. Among the 5 carcinomas associated with exophytic papillomas, HPV was demonstrated together with p53 overexpression in 3 cases (60%). In addition, one case more was with p53 overexpression. CONCLUSION: An inverse relation between HPV and p53 overexpression in sinonasal carcinomas associated with inverted papillomas appears to have been demonstrated. HPV and p53 might also have an etiological role among the carcinomas associated with exophytic papillomas.     

Molecular transformation of recurrent respiratory papillomatosis: viral typing and p53 overexpression

Recurrent respiratory papillomatosis (RRP) is a histologically benign disease of the larynx, trachea, and bronchi. Here we report on the histologic and molecular characteristics of 7 cases of malignant transformation of RRP to squamous cell carcinoma (SCCA). The clinical histories of 7 patients with RRP who developed SCCA were carefully reviewed. Sequential biopsies were available from 5 of the 7 cases of spontaneous transformation of RRP to SCCA and were reviewed. In addition, p53 protein overexpression and human papillomavirus (HPV) typing for all cases was examined. The average age of patients with juvenile-onset RRP was 3 years, and that of patients with adult-onset RRP was 31 years. The average age of onset of transformation to SCCA was 28 years. All patients had laryngeal involvement with RRP, and 3 of the 7 patients had tracheal extension of disease. Five patients were tracheotomy-dependent. Four of the 7 patients developed SCCA of the lung, while 3 patients developed laryngeal SCCA. There was no consistent histologic progression from squamous papilloma to papilloma with dysplasia, and all but 1 of the SCCAs were well differentiated. The overexpression of p53 protein was variable in each of the 5 patients. We detected HPV types 6/11 in papillomas from 3 patients, and HPV types 6/11, 16/18, and 31/33/51 in a papilloma of a fourth patient. No HPV DNA was detected in papillomas of 2 patients. We found HPV 6/11 in 4 of the carcinomas. We conclude that the spontaneous transformation of RRP to SCCA is not characterized by a histologic progression through dysplasia over time. Transformation can result in the loss of HPV expression. It does not appear that p53 is a molecular marker for monitoring the transformation process. Thus, these cancers may be very difficult to diagnose histologically and clinically early in the course of the transformation of the disease.     

喉乳头状瘤临床行为与细胞增殖活性关系的研究

利用ISH方法以及免疫病理手段对不同型的LP组织的标本进行PCNA、P53检测，探讨不同型HPV与LP的发生发展的相关机制，以寻求有效的检测方法帮助临床对LP的预后进行评估。标本选自1994年1月～1995年12月间我院收治的LP共36例。ISH法HPV6b／11阳性率为75％，明显高于HPV16和／或HPV18的表达。10例喉鳞癌各有1例HPV16、18阳性，无HPV6b／11阳性。ABC法行P53检测，36例LP标本中仅1例恶变组织阳性表达Ⅱ级（2．8％）；10例喉鳞癌中9例阳性表达（90．0％），其中Ⅱ级以上阳性表达6例（60．0％）。PCNA阳性表达27／36例（75％）；其中JOP组与AOP组阳性率有显著差异（P＜0．05）。本研究表明LP与HPV感染有极为密切的关系，认为HPV分型的检测在判断LP转归中有意义。PCNA阳性表达程度在预测LP肿瘤的活跃程度方面是一个很有意义的指标。P53蛋白表达在喉鳞癌与LP中有显著差异。     

Human papilloma virus infection and expression of p16 protein in laryngeal papilloma and laryngeal carcinoma]

OBJECTIVE: To evaluate the role of human papilloma virus (HPV) infection and inactivation of p16 gene in laryngeal papilloma (LP) and laryngeal squamous cell carcinoma (LC). METHODS: HPV consensus primers direct in situ polymerase chain reaction (ISPCR) and immunohistochemical method were applied to detect the presence of HPV genomes (1, 6, 8, 11, 13, 16, 18, 30, 31, 32, 33, 45, 51) and the expression of p16 protein respectively in 93 cases of formalin-fixed, paraffin-imbedded specimens, which contained 46 cases of LPs [adult-onset laryngeal papilloma (ALP) 21, juvenile-onset laryngeal papilloma (JLP)25], 26 cases of LCs, 6 cases of normal tissues adjacent to carcinoma, and 15 cases of vocal noduli. RESULTS: (1) The difference of positive rates of HPV-DNA in JLP group (84%, 21/25) and other groups were statistically significant (chi 2 test, P < 0.05). The difference of positive rates of HPV-DNA in ALPs(38.1%, 8/21), in LCs(19.2%, 5/26), in vocal noduli(0%, 0/15), and in normal tissues adjacent to carcinoma(0%, 0/6) were not significant statistically (chi 2 test or Fisher's exact probability test, P > 0.05). (2) The positive rates of expression of p16 protein in ALP group(57.1%, 12/21) and LC group(38.5%, 10/26) were significantly lower than that in vocal nodule group(93.3%, 14/15), in JLP group(88%, 22/25), and in normal tissues adjacent to carcinoma group (100%, 6/6) (chi 2 test or Fisher's exact probability test, P > 0.05). There were no significant differences of positive rates of expression of p16 protein between ALP group and LC group, and between JLP group and vocal nodule group (chi 2 test, P > 0.05). (3) In LPs, the difference of positive rates of p16 protein expression between HPV positive cases and HPV negative cases was significant statistically (chi 2 test, P < 0.05). In LCs, there was no difference in p16 protein expression rate between the two teams(Fisher exact probability test, P > 0.05). CONCLUSION: The pathogenesis of JLP is closely associated with HPV infection and not associated with the inactivation of p16 gene. Conversely, the pathogenesis of ALP and LC is associated with the inactivation of p16 gene and not associated with the HPV infection.     

Coinfection of HPV-11 and HPV-16 in a Case of Laryngeal Squamous Papillomas With Severe Dysplasia

Human papillomavirus (HPV) types 6 and 11 have been associated with benign laryngeal papilloma, while HPV-16 is occasionally associated with laryngeal carcinoma. In this study, a case of laryngeal squamous papillomas with severe dysplasia was evaluated for the presence of HPV infection. The biopsy specimens were taken from a 58-year-old female patient at two different time points 3 months apart. Architecturally, the tumor showed papillary configuration reminiscent of squamous papilloma. Cytologically, the lesion showed morphologic features characteristic of severe squamous epithelial dysplasia. HPV infection was determined by DNA in situ hybridization using type-specific HPV-DNA probes. HPV-11 probes demonstrated homogeneous nuclear staining, suggesting productive viral replication. In contrast, HPV-16 probe produced a speckled pattern, suggesting HPV-16 DNA integration. Normal laryngeal epithelium did not yield specific hybridization. The presence of HPV-11 and HPV-16 was confirmed by PCR using HPV type-specific primers. Immunocytochemical staining was performed to detect Ki-67, a proliferation marker, and p53. Ki-67 expression was demonstrated throughout the whole thickness of epithelium. Staining for p53 was negative. This study suggests that multiple HPV infections can occur in the same lesion and that HPV-16 infection and its DNA integration may contribute to the occurrence of severe dysplasia in the lesion described.     

喉癌和喉乳头状瘤组织中人乳头状瘤病毒和p16蛋白的检测

目的探讨人类乳头状瘤病毒（humanpapillomavirus,HPV）感染和抑癌基因p16的失活与喉癌和喉乳头状瘤（laryngealpapilloma,LP）发生的相关性,以进一步阐明喉癌和LP的病因和发病机理。方法收集LP46例,其中成人型喉乳头状瘤（adult-onsetLP,ALP）21例,青少年型喉乳头状瘤（juvenile-onsetLP,JLP）25例、喉癌26例、癌旁正常组织6例、声带小结15例,用标记的HPV1,6,8,11,13,16,18,30,31,32,33,45,51通用引物直接法原位聚合酶链反应（polymerasechainreaction,PCR）方法和免疫组化（SP法）方法分别检测HPV-DNA和p16蛋白。结果①HPV阳性率JLP组（84%,21/25）显著高于ALP组（38.1%,8/21）、喉癌组（19.2%,5/26）、声带小结组（0/15）和癌旁组织组（0/6）（χ     

Detection of human papillomavirus in laryngeal squamous dysplasia and carcinoma. An in situ hybridization and signal amplification study

We examined the presence of human papillomavirus (HPV) DNA in 65 cases of laryngeal squamous dysplasia and carcinomas using in situ hybridization with signal amplification in paraffin sections. Hybridization was performed with biotinylated DNA probes for HPV 6/11, 16/18, 31/33 and wide-spectrum HPV (6, 11, 16, 30, 31, 45, 51 and 52). HPV DNA was found in 7 cases of the total sample (10.7%); it was also found in 4 out of 45 (8.8%) cases of invasive carcinoma and in 5 out of 33 (15.5%) cases of squamous dysplasia. Morphological signs suggestive of HPV infection were observed in 35.5% of our sample but they were not related to HPV DNA positivity. In conclusion, HPV probably plays little, if any, role in laryngeal carcinogenesis among the population studied.     

Short-fragment PCR assay for highly sensitive broad-spectrum detection of human papillomaviruses in laryngeal squamous cell carcinoma and normal mucosa: clinico-pathological evaluation

The aim of the study was to determine the prevalence and genotypes of HPV infection in laryngeal cancer specimens, normal mucosa obtained from the surgical margin and laryngeal nodules using a novel high sensitive and specific SPF₁₀ HPV DNA test, PCR/DEIA method and INNO-LiPA genotyping assay. The correlation between HPV presence and clinico-pathological features was analyzed. Tissue samples were collected from 93 primary laryngeal squamous cell carcinoma (LSCC), 49 specimens of normal mucosa and from 22 specimens of laryngeal nodules serving as control group. HPV DNA was amplified by the short PCR fragment (SPF₁₀) primer set using HPV DNA enzyme immunoassay (DNA/DEIA) method and INNO-LiPA HPV genotyping assay. Human papillomavirus was detected in 33 (35.5%) of the 93 samples from LSCC, in 4 (8.2%) of 49 samples of the normal mucosa and it was not detected in any of the sample from the control group. Twenty-eight of 33 (81.8%) were positive for HPV-16, 6 of 33 (18.2%) were positive for HPV-18 and 5 of 33 (15.1%) were positive for HPV-33. Multiple infection was found in 5 of 33 (15.1%); 3 samples were positive for HPV-16 and HPV-33, 2 samples for HPV-16 and HPV-18. There was a statistically significant correlation between the presence of HPV in LSCC tumors and in control group samples and between the presence of HPV in the tumors and normal mucosa from the free surgical margin. The presence of HPV infection in 35.5% of the cases suggests a possible role in the etiology of laryngeal cancer and supports the role of high-risk types of HPV (16, 18 and 33) in LSCC. HPV infection is not likely to influence survival rates as an independent prognostic factor in patients with laryngeal cancer.