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     

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。     

原位杂交在成年和幼年型喉乳头状瘤HPV病因学的对比研究

探讨成年和幼年型喉乳头状瘤ＨＰＶ感染发病差异及其影响因素。方法：用地高辛配基（Ｄｉｇｏｘｉｇｅｎｉｎ）标记ＨＰＶ６和ＨＰＶ１１型作探针，原位核酸杂交方法在２９例成年型喉乳头状瘤（ＡＬＰ）和２１例幼年型喉乳头状瘤（ＪＬＰ）石蜡包埋标本检测ＨＰＶ同源序列。结果：ＡＬＰＨＰＶ６和ＨＰＶ１１阳性率分别为４１４％（１２／２９）和４８３％（１４／２９）；ＪＬＰＨＰＶ６及ＨＰＶ１１阳性率均为７６２％（１６／２１）。ｘ２统计示：两型喉乳头状瘤ＨＰＶ６及ＨＰＶ１１阳性率明显不同（ＨＰＶ６ｘ２＝５９９，ＨＰＶ１１ｘ２＝３９５，Ｐ均小于００５）。结论：１）ＡＬＰ和ＪＬＰＨＰＶ感染发病存在差异。２）ＡＬＰ除了ＨＰＶ感染外，其促发因素不可忽视，ＪＬＰ更倾向于依赖ＨＰＶ感染而发病。     

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。     

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     

Pharyngeal squamous cell papilloma in adult Japanese: comparison with laryngeal papilloma in clinical manifestations and HPV infection

A number of reports have investigated the relationship between laryngeal papilloma and human papilloma virus (HPV) infection. On the other hand, it is unclear whether the HPV infection is involved in the occurrence of pharyngeal papilloma. We hypothesized that HPV infection was involved in the occurrence of pharyngeal papilloma similarly to laryngeal papilloma. To verify this hypothesis, we investigated the presence of HPV infection. Furthermore, clinical manifestations of pharyngeal papilloma, which had rarely been reported, were discussed. A male-to-female ratio, solitary or multiple occurrences, and koilocytosis were examined in cases with pharyngeal papilloma. HPV DNA was examined with unfixed surgically resected specimens of pharyngeal papilloma. A screening test by the liquid-phase hybridization method was carried out for the HPV high-risk group (16, 18, 31, 33, 35, 39, 45, 51, 56, 58, 59, and 68) and HPV low-risk group (6, 11, 42, 43, 44). As a control, 15 cases with laryngeal papilloma for which the same screening test was carried out were employed. Pharyngeal papilloma occurred as a solitary lesion more often, whereas laryngeal papilloma occurred as multiple tumors more frequently. The HPV infection rate was 0% in pharyngeal papilloma cases, which was in stark contrast with 66.7% in the HPV low-risk group in laryngeal papilloma cases. Pharyngeal papilloma occurred as a solitary lesion in females more frequently. Contrary to our hypothesis, the involvement of HPV infection was unlikely in the occurrence of pharyngeal papilloma.     

Effect of human papilloma virus expression on clinical course of laryngeal papilloma

Conclusion. Our observations suggest that human papilloma virus (HPV) 6/11 is the main causative agent of laryngeal papilloma and that detection of active HPV DNA expression may be helpful in identifying patients with aggressive recurrent laryngeal papilloma. Objectives. HPV is assumed to be the main causative agent of this disease. We investigated the expression of the entire genotype of HPV in cases of laryngeal papilloma and correlated their expression with the clinical course of the disease. Subjects and methods. Seventy cases of laryngeal papilloma were evaluated for the presence of the HPV genome by in situ hybridization (ISH) using wide-spectrum HPV DNA probe. Specific types of HPV infection were determined by DNA ISH using type-specific HPV DNA probes (HPV 6, 11, 16, 18, 31, 33). Separate analyses were conducted comparing viral types, frequency of recurrences and duration of disease-free periods. Results. We detected HPV DNA in 40 of the 70 laryngeal papilloma cases (57%). In particular, HPV DNA was detected in 75% of the juvenile types. There were significant associations between HPV and laryngeal papilloma (p<0.01). Among the HPV-positive cases, major specific types were HPV 6/11 (97%). Significant associations were also noted between viral expression and clinical course.     

Effect of human papilloma virus expression on clinical course of laryngeal papilloma

Conclusion. Our observations suggest that human papilloma virus (HPV) 6/11 is the main causative agent of laryngeal papilloma and that detection of active HPV DNA expression may be helpful in identifying patients with aggressive recurrent laryngeal papilloma. Objectives. HPV is assumed to be the main causative agent of this disease. We investigated the expression of the entire genotype of HPV in cases of laryngeal papilloma and correlated their expression with the clinical course of the disease. Subjects and methods. Seventy cases of laryngeal papilloma were evaluated for the presence of the HPV genome by in situ hybridization (ISH) using wide-spectrum HPV DNA probe. Specific types of HPV infection were determined by DNA ISH using type-specific HPV DNA probes (HPV 6, 11, 16, 18, 31, 33). Separate analyses were conducted comparing viral types, frequency of recurrences and duration of disease-free periods. Results. We detected HPV DNA in 40 of the 70 laryngeal papilloma cases (57%). In particular, HPV DNA was detected in 75% of the juvenile types. There were significant associations between HPV and laryngeal papilloma (p<0.01). Among the HPV-positive cases, major specific types were HPV 6/11 (97%). Significant associations were also noted between viral expression and clinical course.     

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     

Survivin在喉鳞状细胞癌、喉乳头状瘤及喉黏膜白斑中的表达及意义

目的 探讨Survivin蛋白在喉鳞状细胞癌、喉乳头状瘤及喉黏膜白斑中的表达及其意义。方法 应用免疫组化对46例喉鳞状细胞癌、24例癌旁组织、20例喉乳头状瘤、28例喉黏膜白斑和16例正常喉黏膜中Survivin的表达情况进行检测。结果 Survivin蛋白在喉癌组织和癌旁组织、喉黏膜白斑及喉乳头状瘤中的阳性表达率分别为71．74％（33／46）、33．33％（8／24）、46．43％（13／28）；40％（8／20），在正常黏膜组织中无表达。喉癌组中的阳性表达率显著高于癌旁组织、喉黏膜白斑、喉乳头状瘤及正常黏膜组织中的表达率。且喉乳头状瘤组及喉黏膜白斑组中发生恶变的组织中Survivin蛋白均为阳性表达。Survivin蛋白的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无关。结论 Survivin在喉癌组织中过度表达参与了喉癌的形成，对喉癌的癌前病变发展成恶性肿瘤的潜在可能性有预测作用。     

人乳头状瘤病毒与喉癌,喉内翻性乳头状瘤发病的关系

应用聚合酶链反应（ＰＣＲ）法检测人乳头状瘤病毒（ＨＰＶ）抗原，并在透射电镜下查找ＨＰＶ颗粒，在１３例喉内翻性乳头状瘤的标本中，有７例找到ＨＰＶ颗粒，８例ＨＰＶ抗原阳性；其中３例喉内翻性乳头瘤恶变的标本中２例找到ＨＰＶ颗粒且抗原阳性，证明喉内翻性乳头状瘤发病及其癌变与ＨＰＶ密切相关。     

微管相关蛋白1轻链3在喉癌中的表达及其意义

目的:探讨微管相关蛋白1轻链3(LC3)在喉癌中的表达及其意义.方法:采用免疫组织化学染色方法检测LC3蛋白在50例喉癌组织、45例癌旁组织、10例喉乳头状瘤及16例声带息肉中的表达情况.采用RT-PCR法检测LC3 mRNA和蛋白在41例喉癌组织、41例癌旁组织及11例声带息肉组织中的表达,并结合临床病理因素进行分析.结果:免疫组织化学结果显示LC3蛋白在喉癌组织、癌旁组织、喉乳头状瘤、声带息肉中的阳性率分别为60.0%、93.3%、90.0%、93.8%.LC3在喉癌组织中的阳性表达率明显低于癌旁及声带息肉组织(χ2=18.135,P＜0.01).RT-PCR检测结果显示LC3 mRNA在喉癌组织中相对表达量为0.57±0.08,与癌旁组织(0.99±0.11)及声带息肉组织(1.07±0.05)中的相对表达量比较差异亦有统计学意义(F=255.872,P＜0.01).LC3在喉癌中的蛋白表达与肿瘤部位、病理学分级有关(P＜0.05),与患者的年龄、T分期、临床分期及淋巴结转移无关(P＞0.05).结论:LC3在喉癌中的表达下调,自噬活性的改变可能与喉癌发生、发展有关.     

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.     

Survivin与PTEN在喉部鳞状细胞癌变中的表达关系初探

目的:研究声带息肉、成人型喉乳头状瘤和喉鳞状细胞癌3种疾病中Survivin和PTEN的表达特点,以了解喉鳞状细胞从增生到癌变的可能机制。方法:选取符合临床与病理诊断的声带息肉患者18例、成人型喉乳头状瘤患者10例和喉鳞状细胞癌患者18例,将每个患者的病理蜡块标本各连续切片2张,分别进行Sur-vivin和PTEN免疫组织化学检测。比较致癌基因Survivin和抑癌基因PTEN在这3种疾病同一区域鳞状上皮细胞中的表达差异。结果:声带息肉的Survivin和PTEN阳性率均显著低于喉乳头状瘤和喉鳞状细胞癌(P<0.05),而喉乳头状瘤的Survivin和PTEN阳性率均低于喉鳞状细胞癌,但均没有显著性差异(P>0.05),Sur-vivin和PTEN形成近似声带息肉<成人型喉乳头状瘤<喉鳞状细胞癌的表达趋势。在声带息肉、喉乳头状瘤及喉鳞状细胞癌3种疾病中,PTEN的阳性率均显著低于Survivin(P<0.05)。结论:Survivin和PTEN在喉良性增生性疾病的鳞状细胞中部分表达,而在良性和恶性肿瘤中过度表达,体现出随恶性程度增加而逐渐升高的表达趋势,Survivin和PTEN虽然功能相反,但作用机制不同,非对立消长关系,PTEN体现出一定的竞争表达特点,并从属于Survivin的表达,两者的统一部分决定了喉鳞状细胞的恶变程度。     

喉癌组织Rb、P53、C-myc和HPV基因测定及意义

采用PCR技术检测了33例喉癌及4例正常喉组织Rb、P53、C-myc、HPV基因的异常状况，结果：P53基因突变率为69.69%(23/33)(P＜0.01)，Rb基因突变率为48.48%(16/33)(P＜0.01)，C-myc基因扩增率为58.62%(17/29)(P＜0.01)，HPV阳性率为72.72%(24/33)(P＜0.01)。结合病毒感染和重度吸烟探讨发生喉癌因素的相互关系，研究表明：HPV感染和吸烟与C-myc基因激活和Rb、P53基因突变失活有关，是导致喉癌发生发展的多因素、多基因、多阶段的相互作用。     

一氧化氮合酶(NOS)在喉鳞癌中的表达及意义

目的：检测喉鳞状细胞癌组织中一氧化氮合酶（nitricoxidesynthase,NOS）的表达,并分析其表达与喉鳞癌病理特性的关系。方法：应用免疫组织化学技术对42例喉鳞癌,15例喉乳头状瘤,5例声带息肉组织中的iNOS,eNOS表达情况进行对比。结果：喉鳞癌中iNOS的阳性表达率为83．3％（35／42）,喉乳头状瘤阳性表达率为40．0％（6／15）,二者比较有显著性差异（x2＝9．47,P＜0．05）;eNOS在喉鳞癌组阳性表达为52．4％（22／42）,喉乳头状瘤组为6．7％（1／15）,二者比较有显著性差异（x2＝9．59,P＜0．05）;iNOS和eNOS的表达与喉鳞癌的临床分期无显著相关性（P＞0．05）。iNOS在低分化癌表达率为86．7％（13／15）,在高分化癌为75．0％（12／16）,但iNOS强阳性表达在高分化型鳞癌中为12.6％（2／16）,在低分化鳞癌中为53．3％（8／15）,其差异显著;eNOS在低分化和高分化癌中的表达率分别为73．3％（11／15）和31．3％（5／16）,其差异均有显著性（P＜0．05）。结论：NOS在喉鳞癌有较高的表达,可能为肿瘤细胞间的异常介质信号,与喉癌的发生和分化有关,肿瘤组织内有新生血管形成,其血流由一氧化氮（nitricoxide,ON）调节,明确其作用机理,将可能为喉癌的生物治疗提供理论依据。     

High incidence of malignant transformation of laryngeal papilloma in Taiwan

OBJECTIVES: Papillomas of the larynx include solitary laryngeal papilloma and recurrent respiratory papillomatosis. This study investigated the incidence of malignant transformation and assessed possible risk factors for laryngeal papillomas. STUDY DESIGN: A prospective, longitudinal study. METHODS: Twenty-six consecutive laryngeal papilloma patients were prospectively studied for 5 or more years, and each patient was periodically examined at 3 to 6 month intervals. A detailed epidemiologic questionnaire was administered at the initial visit. After enrollment, tissue obtained during each laryngeal surgery was examined by polymerase chain reaction assay for human papilloma virus (HPV) and typing. RESULTS: During 237 person-years of follow-up, six new, pathologically confirmed cases of laryngeal carcinoma were ascertained (incidence 2.5/100 person-years), and all were associated with HPV-6 or HPV-11. Malignant transformation revealed no correlation with the following: age less than 3 years at diagnosis, sex, history of tobacco use, history of alcohol consumption, family history of laryngeal cancer, or type of laryngeal papilloma. Laryngeal papilloma without demonstrable HPV DNA was the only significant risk factor for malignant transformation (P < .05). The cumulative risk of malignant transformation in subjects without demonstrable HPV DNA was significantly higher than that in HPV-positive patients (relative risk, 8.0; 95% confidence interval, 1.1-60.3; P = .05). CONCLUSIONS: A relatively high incidence of malignant transformation of laryngeal papilloma was noted in Taiwanese patients. Patients without demonstrable HPV DNA require more frequent follow-up and may benefit from anti-HPV vaccinations.     

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。     

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。     

The detection and significance of human papilloma virus 11b virus like particles and its serum antibody in juvenile larynx papilloma]

OBJECTIVE: To study the relationship between human papilloma virus 11b virus like particles (HPV11bVLPS) serum antibody and the development and prognosis of juvenile larynx papilloma (JLP). METHODS: Enzyme linked immunosorbent assay (ELISA) was used to detect the serum HPV11bVLP antibody (Ab) of 46 JLP's samples in different stage and 20 controls using HPV11bVLPS which was produced by recombinant bacilovirus in insect cells. Grouping: A: control group (n = 20); B: the time of onset was 1 years (n = 15); C: the time of onset was 2 years (n = 15); the patients were followed-up 1 year without recurrence (n = 8); E: The patients were followed-up 2 years without recurrence (n = 8). RESULTS: A value of HPV11bVLP Ab among A, B, C, D, E. group were: (0.073 +/- 0.035); (0.120 +/- 0.049); (0.137 +/- 0.057); (0.518 +/- 0.122); (0.557 +/- 0.144). There was a significant difference between JLP patients and the control group (P < 0.05). The level of HPV11bVLP Ab in (D + E) group (0.534 +/- 0.132) was higher than (B + C) group (0.128 +/- 0.053) (t = 14.90, P < 0.001). CONCLUSION: The results suggested that HPV serum antibody was produced in JLP with HPV infection. There is close relationship between the development and prognosis of the disease and the level of HPV11Ab in serum. The assay of serum HPV11bVLPAb and HPV-VLP could be used as immunological study of HPV11-infection associated disease.