小儿喉乳头状瘤HPV-DNA及体液免疫检测

目的：探讨小儿喉乳头状瘤（ＪＬＰ）人乳头瘤病毒（ＨＰＶ）感染途径及发病机理。方法：采用ＰＣＲ及ＰＣＲ产物斑点杂交技术检测ＪＬＰ组织ＨＰＶ－ＤＮＡ;散射免疫比浊法测定血清Ｉｇ及补体Ｃ３。结果：ＪＬＰ组织ＨＰＶ总感染率为９５％（１９／２０）,其中ＨＰＶ。型为５５％（１１／２０）,ＨＰＶ１１为３０％（６／２０）,ＨＰＶ６＋１１型为１０％（２／２０）;ＪＬＰ患者血清ＩｇＧ、ＩｇＡ、ＩｇＭ、Ｃ３值正常,对照     

上呼吸道乳头状瘤HPVDNA的检测

应用多重引物ＰＣＲ技术对４７例复发性呼吸道乳头状瘤病（ＲＲＰ）和９例声带息肉石蜡包埋组织中ＨＰＶ６／１１、１６、１８ＤＮＡ进行了检测。结果发现：①ＲＲＰ组织中ＨＰＶ６／１１ＤＮＡ的阳性率为６８．１％（３２／４７），其中喉乳头状瘤、口咽乳头状瘤、鼻腔和鼻前庭乳头状瘤组织中ＨＰＶ６／１１ＤＮＡ的阳性率分别为７０．４％（１９／２７）、６６．７％（４／６）和６４．３％（９／１４）；②所有ＲＲＰ标本中均未发现特异性的ＨＰＶ１６和ＨＰＶ１８ＤＮＡ；③９例声带息肉标本中四种型号的ＨＰＶＤＮＡ全部阴性。实验结果表明，ＲＲＰ的发病与ＨＰＶ６／１１感染密切相关。多重引物ＰＣＲ检测ＨＰＶＤＮＡ具有敏感、特异、快速、简便等优点，适合于临床广泛开展和进行流行病学调查。     

国人喉癌与人类乳头状瘤病毒相关性的研究

本项研究对１２４例喉不同病变的新鲜组织标本，采用共同引物和多重引物ＰＣＲ的方法，进行ＨＰＶＤＮＡ检测。用共同引物ＰＣＲ检测ＨＰＶ＿（６，１１，１６，１８，３１，３３，３５，４２，５８）九个型别的感染，阳性病例再用多重引物ＰＣＲ进一步分型。结果，①喉癌组：ＨＰＶ感染的总阳性率为４９．１％，其中ＨＰＶ＿（１８）型阳性率１５．８％，ＨＰＶ＿（１６）型为１２．３％，ＨＰＶ＿（１６）和＿（１８）双重型感染为５．３％，ＨＰＶ＿（６／１１）和＿（１８）型混合感染为３．５％，其它型为１２．３％。②颈转移淋巴结组：总阳性率为２１．４％，其中ＨＰＶ＿（１６）、ＨＰＶ＿（１８）及ＨＰＶ＿（１６）和＿（１８）双重型感染各为７．１％。③癌前病变组：ＨＰＶ感染阳性率为１１．１％，为ＨＰＶ＿（６／１１）和＿（１８）型混合感染。④声带息肉组：阳性率７．１％，为ＨＰＶ＿（６／１１）型感染。⑤癌旁及癌周正常喉组织：均为ＨＰＶＤＮＡ阴性。本文对ＨＰＶ在喉癌中致病作用进行了讨论。     

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Ｐ     

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、３３５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５４．２％（１９／３５）,ＨＰＶ１１型感染率为２５．７％（９／３５）,多重型别ＨＰＶ６＋１１感染率为１１．     

人咽喉部良恶性肿瘤与乳头状瘤病毒关系的研究

采用免疫组化及ＤＮＡ斑点杂交技术检测人咽喉部乳头状瘤及鳞状细胞癌组织中人乳头状瘤病毒（ＨＰＶ）壳蛋白抗原及ＨＰＶ６、１１、１６、１８型ＤＮＡ。１１例乳头状瘤ＨＰＶ抗原与ＨＰＶ ＤＮＡ阳性率均为４５．５％。２２例鳞状细胞癌ＨＰＶ抗原阳性率２２．７％，ＨＰＶ ＤＮＡ阳性率２７．３％。乳头状瘤ＨＰＶ检出率与组织学检查的结果相符。提示咽喉部乳头状瘤及鳞状细胞癌的发生、发展与ＨＰＶ感染有关。     

生物素探针原位核酸杂交检测喉乳头状瘤人乳…

采用定位准确、敏感性高、特异性强的原位核酸杂交技术，生物素标记ＨＰＶ６、１１、１６、３０型作探针，在喉乳头状瘤石蜡包埋及冰冻标本检测人乳头瘤病毒同源列。４５例石蜡标本１７例ＨＰＶ６阳性１９例ＨＰＶ１１阳性，ＨＰＶ１６、３０无一例阳性。实验结果为喉乳头状瘤的病毒病因不说提供了直接证据。对喉乳头状瘤发病与ＨＰＶ型别关系，ＨＰＶ型别在幼年型及成年型喉乳头状瘤检出率不同进行了比较和讨论。     

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：     

PCR和PCR产物斑占杂交技术检测成人咽喉病变中人乳头状？…

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５５例咽喉不同病变的新鲜组织标本进行ＨＰＶ６,１１,１６,１８,３３共５型ＨＰＶ－ＤＮＡ感染的检测。结果：在咽乳状瘤组ＨＰＶ感染率为６０％（６／１０）,喉乳头状瘤组为７０％（７／１０）,喉鳞状上皮非典型增生组为２０％（１／５）,声带息肉组为２０％（１／５）,喉癌组为２０％（１／５     

鼻腔鼻窦内翻性乳头状瘤人类乳头状瘤病毒DNA检测

为了解鼻腔鼻窦内翻性乳头状瘤与人类乳头状瘤病毒（ＨＰＶ）之间的关系，采用聚合酶链反应，对３８例患者的４４个鼻腔鼻窦内翻性乳头状瘤的病理组织蜡块进行ＨＰＶ－ＤＮＡ的检测。结果显示，３８例中３０例患者的３０个瘤组织蜡块呈ＨＰＶ阳性，总感染率为６８．２％，其中３０例次ＨＰＶ１１型阳性（６８．２％），１８例次ＨＰＶ１６型阳性（４０．９％），２例次ＨＰＶ１８型阳性（４．５％），其中１８例ＨＰＶ１１，１６、２例ＨＰＶ１１，１８呈双重感染。试验表明，ＨＰＶ与鼻腔鼻窦内翻性乳头状瘤在病因上有一定的相关性。     

HPV11对小儿喉乳头状瘤预后的影响

目的 :研究人乳头状瘤病毒 (HPV)型别对小儿喉乳头状瘤 (JLP)预后的影响。方法 :应用聚合酶链反应结合斑点杂交技术对 2 5例JLP的石蜡标本进行HPV定型分析 ,并统计HPV11、HPV6 感染组的气管切开率和术后复发率。结果 :HPV总检出率为 96.0 % ,其中HPV11为 5 6.0 % ,HPV6 为 4 0 .0 % ,HPV16、18、33无一例阳性。HPV11感染组的气管切开率为 71.4 % ,术后复发率为 85 .7% ;HPV6 感染组的气管切开率为 3 0 .0 % ,术后复发率为4 0 .0 %。两组分别比较 ,其差异均有显著性意义 (P <0 .0 5 )。结论 :HPV6、11与JLP发生密切相关 ,HPV11感染与JLP的喉梗阻和术后复发率相关 ,HPV11感染可作为JLP预后评判的重要依据。     

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     

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.     

The presence of human papillomavirus (HPV) in solitary adult laryngeal papillomas demonstrated by in-situ DNA hybridization with sulphonated probes

Human papilloma virus (HPV) types 6 and 11 have been repeatedly demonstrated in multiple laryngeal papillomas, and there is little doubt that these lesions are caused by HPV. It has been clearly demonstrated in recent reports that the clinical course of solitary adult onset laryngeal papillomas is entirely different from that of multiple papillomas of juvenile as well as of adult onset. We here report the presence of HPV types 6 and 11 in 19 out of 20 solitary papillomas from 16 patients, while HPV types 16 and 18 were totally absent. We conclude that the milder clinical course in such patients is most likely to be due to host factors, rather than to viral factors.     

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。     

HPV6b病毒样颗粒免疫治疗儿童喉乳头状瘤临床初步研究

目的 :研究HPV6bL1病毒样颗粒 (VLP)治疗儿童喉乳头状瘤 (JLP)的安全性和免疫原性。方法 :应用基因工程制备的HPV6bL1VLP 5、10、2 5 μg 3种剂量递增方法对 10例严重复发性JLP患儿进行免疫接种 ,记录不良反应及行血、尿常规和生化检测 ,ELISA法检测血清特异性HPV6bL1VLP抗体 ,对 7例患儿进行迟发性超敏反应 (DTH)试验 ,纤维喉镜随访观察喉部病变情况。结果 :接种后患儿无局部和全身不良反应 ,血清均能产生特异性的中和抗体 ,接种前 3天和 3种剂量完成后及开始治疗 1年后的血清抗体吸收度A均值分别为 0 .110± 0 .0 35 ,0 .310± 0 .0 12 ,0 .5 87± 0 .0 12 ,0 .75 2± 0 .0 19,0 .772± 0 .0 13。第 1剂量完成后与接种前 3天A均值比较 ,第 2剂量与第 1剂量完成后比较 ,第 3剂量与第 2剂量完成后比较 ,接种 1年后与对照组比较 ,各组间差异均有统计学意义 (均P <0 .0 1)。 7例行DTH试验的患儿均呈阳性反应。经免疫治疗后的 10例患儿未见复发。结论 :HPV6bL1VLP对JLP具有安全性和免疫原性 ,可成为防治JLP的有效疫苗。     

表皮生长因子受体及血管内皮生长因子在喉乳头状瘤中的表达

目的探讨表皮生长因子受体（EGFR）和血管内皮生长因子（VEGF）在喉乳头状瘤（IJP）中的表达及意义。方法采用免疫组化二步法检测10例成人型喉乳头状瘤（ALP）、19例幼年型喉乳头状瘤（JLP）石蜡标本中EGFR、VEGF的表达与分布;并以10例声带息肉作为对照组。结果EGFR和VEGF在ALP、JLP组上皮层的表达水平明显高于对照组（P〈0．05）。EGFR在ALP、JLP表皮组织全层均有强阳性表达,VEGF呈现以基底层、棘层细胞显著表达,到颗粒层表达逐渐减弱的模式。VEGF在ALP、JLP和对照组间质的血管内皮细胞、炎症细胞、成纤维细胞中也有表达,但3组问VEGF的表达元显著统计学差异（P〉0．05）。JLP组上皮中VEGF的表达评分结果（7．133±0．061）比ALP组（6．934±0．041）高,两组比较有统计学意义（P〈0．05）。结论VEGF、EGFR在LP组织中的过度表达可能在LP的上皮细胞过度增生和血管大量形成中发挥重要作用,JLP比ALP具有更强的增殖活性。     

Detection of human papillomavirus genome in nasolaryngeal papillomas using digoxigenin labeled DNA probes

It is being reported that human papillomavirus (HPV) has been implicated in the pathogenesis of various neoplastic lesions of the genital organs. To investigate the etiological role of HPV and its types in nasolaryngeal papillomas, we retrospectively analyzed HPV genomes by nucleic acid hybridization methods; for detecting DNA and mRNA, we employed the recently developed nonradioactive (digoxigenin labeled) DNA probes and compared the results by radioisotope methods. In total, 43 cases of papillomatous lesions were examined. They were verruca vulgaris of the nasal vestibule (Nr = 2), nasal inverted papilloma (IP, Nr = 26), and laryngeal papilloma (Nr = 15). HPV types examined were type 2, 6, 11, 16 and 18. Two cases of verruca vulgaris were shown to contain HPV-2 DNA and its mRNA by in situ hybridization. HPV-11 DNA was detected in 3 cases (12%) of nasal inverted papilloma whereas HPV-16 was detected in 1 case (4%); the latter case was associated with squamous cell carcinoma. These results suggest that HPV may be implicated in the development of IP, and HPV-16 may play an important role in the malignant transformation of IP. In the cases of multiple laryngeal papilloma (Nr = 8, one juvenile type and 7 adult type), either HPV-6 or HPV-11 was detected at the high rate (6/8, 75%). The presence of the HPV genomes provides strong evidence for the HPV etiology of these laryngeal papillomas. Whereas in the cases of adult single laryngeal papilloma (Nr = 7), HPV was not detected. Technically, the sensitivity of digoxigenin (DIG) labeled DNA probe was almost same as 35S labeled probe by dot blot hybridization, thus we applied DIG labeled probe to Southern blot hybridization with low background. By in situ hybridization using digoxigenin labeled probes, the rates of HPV detection were almost equal to those by 35S labeled probes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human papillomavirus in laryngeal papillomas and in adjacent normal epithelium

Eleven adults with laryngeal papillomas were studied for the presence of human papillomavirus (HPV) DNA by in situ hybridization. As well as from the papillomas, three additional biopsies were taken from the normal-appearing mucosa as follows: the involved vocal cord, the opposite vocal cord (when the papilloma was unilateral), and from the ventricular fold on the side of the lesion. These normal tissues were analysed by polymerase chain reaction (PCR) to detect HPV DNA. All except one of the 11 papillomas contained HPV DNA; nine were HPV 6/11 DNA positive and one positive for HPV 16 DNA. The normal-appearing laryngeal mucosa harboured HPV DNA in eight out of 11 patients. The present results strongly support the concept that the adult-type laryngeal papilloma is an HPV-induced lesion, mostly due to HPV types 6 and 11. The persistence of HPV DNA in the adjacent normal epithelium is consistent with the frequent recurrence of these lesions.