非生殖器部位脂溢性角化病组织中HPVL1蛋白及HPV16、18DNA的检测及意义

目的 观察非生殖器部位脂溢性角化病(SK)组织中HPV L1蛋白及HPV16、18 DNA的表达变化,并探讨其意义.方法 分别采用免疫组织化学法、PCR法检测43例SK组织,15例正常皮肤组织中的HPV L1蛋白( HPV1、6、11、16、18、31)及HPV16、1g DNA.结果 SK组织中HPV L1蛋白阳性20例(46.51％),正常皮肤组织中HPV L1蛋白阳性4例(26.67％);两组阳性表达率比较,P＞0.05.SK组织及正常皮肤组织中,均未检测到HPV16、18 DNA.结论 非生殖器部位SK组织中并不存在HPV16、18 DNA,其发病与高危型HPV无关;但是非生殖器部位SK组织中有HPV1、6、11、31中某-型或者某几型L1蛋白的存在,属于非特异性感染.     

灰树花多糖诱导HeLa细胞凋亡的体外实验

目的 观察灰树花多糖(PGF)对HeLa凋亡的影响.方法 采用MTT法研究PGF对HeLa细胞增殖的体外抑制作用,HE染色观察凋亡细胞形态,免疫组化法检测caspase-3蛋白表达.结果 PGF以时间和剂量依赖的方式抑制HeLa细胞的生长,HE染色观察到凋亡细胞的形态学改变,免疫组化检测到caspase-3蛋白高表达.结论 PGF促进HeLa细胞凋亡,其作用机制可能与caspase-3蛋白表达增高有关.     

幽门螺杆菌L型和HPV16,18感染在人食管癌中同步检测

目的研究幽门螺杆菌L型(Hp-L型)和HPV16,18感染与人食管癌的关系,初步探讨Hp-L型致癌的机制.方法对112例食管鳞癌,30例正常食管粘膜,应用革兰染色和免疫组化染色(ABC法)技术检测Hp-L型,两者同时阳性者定为Hp-L型感染阳性.另用免疫组化染色(SP法)检测HPV16,18蛋白.结果革兰染色L型检出阳性率(67.9%)高于正常对照组(26.7%),两者有显著性差异,且与免疫组化染色Hp-L型抗原检出阳性率(65.2%)无显著性差异(P>0.05).Hp-L型检出阳性率为61.6%(69/112).革兰染色油镜(10×100)下观察发现,Hp-L型形态不规则,呈多形性;大小不等;有巨形体、圆球体、原生小体等;大多呈革兰染色阴性(红色),少数呈革兰染色阳性(紫色);可在癌巢、癌间质或粘膜层聚集成堆,也可散在分布于其中,部分癌细胞及粘膜上皮细胞内也见到L型或L型粘附于这些细胞表面.免疫组化染色HPV16,18表达阳性率为69.2%,与Hp-L型检出阳性率无显著性差异(P>0.05),Hp-L型和HPV16,18同时阳性者占59.8%(67/112)结论Hp-L型和HPV16,18感染与食管癌发生密切相关.Hp-L型感染可能是食管癌除病毒之外的又一生物性致癌因素,在食管癌的发生中与HPV16,18具有同样重要的意义     

N-钙黏素融合蛋白对P19细胞神经分化的影响

目的 探讨细胞问黏附分子对胚胎癌P19细胞体外定向神经细胞分化的作用.方法 通过单层培养技术,采用视黄酸为诱导剂诱导P19细胞神经细胞分化,在浓度分别为0.1%明胶、2.5 mg/L和10 mg/L的N-钙黏素融合蛋白(N-cad-Fc)包被的培养板上进行培养,观察细胞形态的变化情况,用神经细胞特异标志蛋白β-Ⅲ型微管蛋白(Tuj-1)抗体进行免疫荧光染色,反转录聚合酶链式反应(RT-PCR)检测干细胞标志蛋白(oct3/4)、神经前体细胞标志蛋白巢蛋白(nestin)、神经元素1(ngn1)、微管相关蛋白(map2)和神经胶质细胞的特异性标志蛋白(gfap)的表达情况.结果 使用5×10-7mol/L的视黄酸成功诱导了P19细胞神经细胞分化,0.1%明胶组、2.5 mg/L N-cad-Fc组和10 mg/L N-cad-Fc组分化为神经元细胞的百分率分别为7.8%、28.6%和26.8%,在包被有N-cad-Fc的培养板上培养,神经标志性蛋白ngn1和map2的表达明显提前、增强,干细胞标志蛋白oct3/4表达提前减弱,未见神经胶质细胞的特异性标志蛋白gfap表达.结论 视黄酸可以诱导P19细胞向神经细胞分化,细胞间黏附分子对分化有促进作用.     

苦参碱诱导大鼠肝卵圆细胞株WB-F 344细胞分化中Wnt-1信号通路的作用

目的: 研究苦参碱干预体外培养的大鼠肝卵圆细胞分化中Wnt-1信号通路的作用.方法: 体外培养大鼠肝卵圆细胞,分别用不同浓度的苦参碱(0.001、0.01、0.05、0.2、0.5、1、1.5、2 g/L)处理卵圆细胞,HE染色观察细胞的形态变化,免疫细胞化学检测细胞内甲胎蛋白、白蛋白、Wnt-1信号蛋白的表达.结果: 苦参碱对肝卵圆细胞的生长有抑制作用,且苦参碱浓度越大,作用时间越长,抑制作用越明显,0.05、0.2、1 g/L的苦参碱作用72 h后,对卵圆细胞的抑制率分别为24.16%±2.03%、40.25%±3.92%和67.31%±6.04%.0.01 g/L的苦参碱即可使肝卵圆细胞的形态发生变化,细胞核变大、变圆,核浆比例减小,双核细胞增多;免疫细胞化学结果显示,0.2 g/L的苦参碱能够使肝卵圆细胞的ALB表达增高,AFP的表达降低,Wnt-1信号蛋白的表达量降低.结论: 苦参碱可以抑制Wnt信号通路的传导,使卵圆细胞向着成熟肝细胞的趋势分化.     

制备HPV L1 VLP的基因工程技术应用进展

人乳头瘤病毒（HPV）感染与宫颈癌的发生密切相关。研究发现，HPV的主要衣壳蛋白L1能够单独或与次要蛋白L2共同在体外自行组装成病毒样颗粒（VLP），该颗粒具有与成熟HPV相同的形态学和免疫学特征，且具有型内高度保守性和型间高度同源性，又有与天然病毒颗粒相似的能诱生中和抗体的抗原表位，且不含病毒DNA，因而成为HPV预防性疫苗研究的重要靶抗原。近年来通过基因工程技术在真核和原核细胞中成功表达了多种型别的HPVL1蛋白，且均可在相应宿主细胞内组装成VLP。现将制备HPV L1 VLP预防疫苗的基因工程技术的应用进展情况综述如下。     

肝细胞蛋白在肠上皮化生型Barrett食管中的诊断价值

目的 比较肝细胞蛋白、细胞角蛋白7、细胞角蛋白20、上皮膜抗原(EMA)4种蛋白在Barrett食管化生的肠上皮中表达的差异。方法 采用免疫组化SP法检测23例肠上皮化生型Barrett食管标本中肝细胞蛋白、细胞角蛋白7、细胞角蛋白20、．EMA四种蛋白的表达，同时进行爱先蓝一高碘酸一无色品红[AB(pH2．5)-PAS]染色标记，比较表达的差异。结果化生的肠上皮中均可见肝细胞蛋白、细胞角蛋白7、细胞角蛋白20及AB(pH2．5)-PAS染色的阳性表达；肝细胞蛋白在所有肠化上皮细胞胞质中表达，胃及肠上皮均不表达；细胞角蛋白20、细胞角蛋白7在肠化上皮细胞胞质、部分胃柱状上皮中呈阳性表达；EMA在小肠及肠化上皮以外的其他上皮均有表达；AB(pH2．5)-PAS染色蓝染部分仅为肠上皮杯细胞中的黏液，其余的胃上皮细胞显示红色，鳞状上皮不显色。结论 肝细胞蛋白可作为特异性标记化生肠上皮的首选指标，细胞角蛋白20、细胞角蛋白7亦是肠上皮化生的敏感指标，但特异性不强；AB(pH2．5)-PAS及EMA在显示肠上皮化生方面不具有优势。     

体外培养的肝癌细胞株与正常肝细胞株蛋白质的差异表达

目的:运用SELDI蛋白质芯片技术分析体外培养的肝癌细胞株(HepG2)与正常肝细胞株(L02)蛋白质表达差异．方法:在体外培养HepG2和L02细胞株,收获细胞,将细胞用细胞裂解液裂解后,采用SELDI蛋白质芯片技术用IMAC3 及WCX2芯片检测HepG2、L02的蛋白质谱．结果:体外培养的肝癌细胞株与正常肝细胞株的蛋白质存在差异表达,IMAC3芯片共捕获61个蛋白,发现差异峰7个,与 L02细胞相比,其中3个差异蛋白在肝癌细胞中高表达,4个差异蛋白在肝癌细胞中低表达．WCX2芯片共捕获91个蛋白, 发现差异峰14个,其中3个差异蛋白在肝癌细胞中高表达,11 个差异蛋白在肝癌细胞中低表达．结论:SELDI蛋白芯片技术检测肝癌细胞株与正常肝细胞株蛋白质的差异表达方法简便,敏感性高,重复性好．     

亚砷酸钠对人皮肤永生化角质形成细胞角化相关基因和核转录因子红系相关因子2mRNA表达的影响

目的 观察不同浓度亚砷酸钠(NaAsO2)对人皮肤永生化角质形成细胞(HaCaT)角化相关基因和核转录因子红系相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)mRNA表达的影响.方法 用0.00(对照)、3.13、6.25、12.50、25.00、50.00、75.00、100.00 μmol/L NaAsO2处理HaCaT细胞48h,采用2-(2-甲氧基-4-硝基苯基)-3-(4-硝基苯基)-5-(2,4-二磺酸苯)-2H-四唑单钠盐(CCK-8)法检测细胞增殖率.根据细胞增殖率检测结果,选择0.00(对照)、6.25、12.50、25.00μmol/L NaAsO2处理HaCaT细胞48 h,采用实时荧光定量PCR法检测HaCaT细胞角蛋白1(Keratin1,K-1)、角蛋白10(Keratin10,K-10)、总苞蛋白(Involurin,Inv)、兜甲蛋白(Loricrin,Lor)和Nr2的mRNA表达.结果 与对照组(100.05％)比较,6.25、12.50、25.00、50.00、75.00、100.00 μmol/L NaAsO2组HaCaT细胞增殖率(83.06％、51.04％、39.52％、24.51％、16.99％、9.04％)明显降低,半数致死量为12.38 μmoL/L.与对照组(1.06±0.28、1.00±0.12、1.00±0.08)比较,6.25、12.50、25.00 μmol/L NaAsO2组HaCaT细胞K-1、Inv、Lor mRNA表达(0.08±0.04、0.13±0.12、0.05±0.03,0.47±0.11、0.21±0.09、0.10±0.15,0.50±0.27、0.31±0.10、0.57±0.23)明显降低(P均＜0.05),但K-10 mRNA表达呈现升高趋势,其中6.25μmol/L NaAsO2组(1.83±0.45)明显高于对照组(1.07±0.14,P＜ 0.05);而12.50、25.00 μmol/L NaAsO2组Nrf2 mRNA表达(0.13±0.07、0.69±0.33)明显低于对照组(1.00±0.09,P均＜0.05).结论 NaAsO2 可通过影响细胞角化相关基因和Nrf-2 mRNA表达,抑制HaCaT细胞增殖与角化.     

A case of esophageal squamous cell carcinoma with positive HPV 11

HPV (human pappiloma virus) infection is an etiologic risk factor for esophageal carcinoma with several studies supporting its carcinogenic role. The main strains are HPV16 and 18 and the less frequent strains 11, 6, 31 and 36. We report the case of a 58 year old male, smoker, admitted to our hospital for progressive dysphagia. Previous endoscopies and biopsies were compatible with a hyperkeratotic esophagus, negative for dysplasia. Second endoscopy showed a hyperkeratotic, verrucous, esophagus with two circumferential stenosis which were dilated with TTS (Through the Scope) balloon. Biopsies revealed multiple squamous cell abnormalities, negative for dysplasia and positive for HPV 11. Symptoms recurred, and he was submitted to another upper gastrointestinal endoscopy with dilation of the stenosis and esophageal stent placement. Two months later, esophagectomy was performed, and the histopathological analysis revealed a squamous cell carcinoma of the esophagus (T2, N0, M0, G1). The patient died after surgery due to acute respiratory distress syndrome.     

Human papillomavirus (HPV) infections and their associations with squamous cell neoplasia

Current data implicating an etiological role of Human papillomavirus (HPV) infections in precancer lesions (intraepithelial neoplasia) and squamous cell carcinoma of both the genital tract and other sites (oral cavity, larynx, skin, esophagus, nasal cavity, bronchus) can be summarized as follows: a) HPV involvement in benign, precancer, and malignant genital squamous cell lesions has been demonstrated by morphological, immunohistochemical and DNA hybridization techniques; b) HPV infections in the genital tract are sexually transmitted (STD) and associated with the same risk factors as development of cervical carcinoma; c) natural history of cervical HPV lesions is equivalent to that of CIN, i.e. they are potentially progressive to carcinoma in situ; d) latent HPV infections exist in the genital tract of both sexes; e) animal models exist, where papillomaviruses induce malignant transformation; f) HPV 11 induces transformation of human squamous epithelium in vivo (nude mouse renal subcapsule assay); g) malignant transformation of HPV lesions seems to depend on virus type and the physical state of its DNA, i.e. whether or not integrated in the host cell DNA; h) malignant transformation most probably requires synergistic actions between HPV and chemical or physical carcinogens or other infectious agents; i) genetic disposition (at least in animals) significantly contributes to malignant transformation; j) immunological defence mechanisms of the host probably are capable of modifying the course of HPV infection (efficacy in man remains to be elucidated). Many details of the molecular mechanisms still remain to be clarified, however. No applicable model systems exist to elucidate these issues, or the mechanisms leading to the progression to invasive cancer. Improved tissue culture systems for in vitro differentiation of keratinocytes should help in elucidating the biology of papillomaviruses and their interaction with cell division and differentiation.     

A Novel In Vitro Culture Model System to Study Merkel Cell Polyomavirus–Associated MCC Using Three-Dimensional Organotypic Raft Equivalents of Human Skin

Merkel cell polyomavirus (MCPyV) is a human polyomavirus causally linked to the development of Merkel cell carcinoma (MCC), an aggressive malignancy that largely arises within the dermis of the skin. In this study, we recapitulate the histopathology of human MCC tumors in vitro using an organotypic (raft) culture system that is traditionally used to recapitulate the dermal and epidermal equivalents of skin in three dimensions (3D). In the optimal culture condition, MCPyV+ MCC cells were embedded in collagen between the epidermal equivalent comprising human keratinocytes and a dermal equivalent containing fibroblasts, resulting in MCC-like lesions arising within the dermal equivalent. The presence and organization of MCC cells within these dermal lesions were characterized through biomarker analyses. Interestingly, co-culture of MCPyV+ MCC together with keratinocytes specifically within the epidermal equivalent of the raft did not reproduce human MCC morphology, nor were any keratinocytes necessary for MCC-like lesions to develop in the dermal equivalent. This 3D tissue culture system provides a novel in vitro platform for studying the role of MCPyV T antigens in MCC oncogenesis, identifying additional factors involved in this process, and for screening potential MCPyV+ MCC therapeutic strategies.     

MSM人群中HIV合并HPV感染及其亚型分析

目的了解男男性行为人群（MSM）艾滋病病毒（HIV）感染者及病人中,合并人乳头状瘤病毒（HPV）感染及其亚型分布状况。方法采用杂交捕获法,分别对MSM人群的HIV感染者及病人（观察组）和男性尖锐湿疣患者（对照组）进行HPV分型检测。结果在观察组91例患者中,合并HPV阳性检出率为73.63%（67/91）,共检出15个HPV亚型,其中高危亚型11个（16、18、31、33、45、52、56、59、66、68、73型）,低危亚型4个（6、11、42、43型）。单一亚型感染占31.34%（21/67）,多重亚型感染占68.66%（46/67）。高危亚型感染占44.78%（30/67）,低危亚型感染占55.22%（37/67）。观察组病例的多重亚型HPV感染率（χ2=30.6474,P〈0.000 1）和高危亚型HPV感染率（χ2=22.3210,P〈0.000 1）都高于对照组,差异有统计学意义。结论在MSM人群中,HIV合并HPV感染呈较高流行状态,HIV感染者罹患肛门生殖器肿瘤的风险增加,提示加强HIV感染者HPV感染的防治有重要意义。     

Vaccine-Relevant Human Papillomavirus (HPV) Infections and Future Acquisition of High-Risk HPV Types in Men

Background.Little is known about type-specific associations between prevalent human papillomavirus (HPV) infections and risk of acquiring other HPV types in men. Data on natural clustering of HPV types are needed as a prevaccine distribution to which postvaccine data can be compared. Methods.Using data from a randomized controlled trial of male circumcision in Kisumu, Kenya, adjusted mean survival ratios were estimated for acquisition of any-HPV, high-risk (HR) HPV, and individual HR-HPV types among men uninfected as compared to those infected with vaccine-relevant HPV types 16, 18, 31, 45, 6, or 11 at baseline. Results.Among 1097 human immunodeficiency virus-negative, uncircumcised men, 2303 incident HPV infections were detected over 2534 person-years of follow-up. Although acquisition of individual HR-HPV types varied by baseline HPV type, there was no clear evidence of shorter times to acquisition among men without vaccine-relevant HPV-16, -18, -31, -45, -6, or -11 infections at baseline, as compared to men who did have these infections at baseline. Conclusions.These prospective data on combinations of HPV infections over time do not suggest the potential for postvaccination HPV type replacement. Future surveillance studies are needed to definitely determine whether elimination of HPV types by vaccination will alter the HPV type distribution in the population.     

干扰素抗性株人类乳头瘤病毒的型别分析

目的检测并分析干扰素抗性株人类乳头瘤病毒的型别。方法于尖锐湿疣局部注射干扰素,在治疗前应用导流杂交技术,对所有的疣体检测人类乳头瘤病毒的型别,并分别对干扰素治疗敏感及抵抗的乳头瘤病毒型别进行统计分析。结果人类乳头瘤病毒6型的治疗抵抗率为9．6％,人类乳头瘤病毒11型的治疗抵抗率却高达31．8％,经统计学分析,差异有统计学意义（Х^2=11．2792,P〈0．05）。结论人类乳头瘤病毒11型较6型更易出现干扰素治疗抵抗。     

Therapeutic vaccines against HPV16-associated tumors. Minireview

Human papilloma viruses (HPV) were found to be closely associated with several types of anogenital tumors, particularly with cervical carcinomas (CC). Of more than 100 HPV types characterized until now, 11 have been classified as high-risk types and detected in human tumor tissue by molecular and immunological techniques. Immunological intervention against HPV can be envisaged at two levels, prophylactic and therapeutic. The therapeutic vaccines constructed to counteract tumors which are already developed utilize two nonstructural early proteins coded by HPV, the products of their E6 and E7 oncogenes. These E6/E7 oncoproteins are the only HPV-coded proteins expressed in CC; they are involved in malignant transformation of HPV-infected cells, their presence is necessary for the maintenance of the malignant phenotype of the cells, and their expression correlates with the transforming potential of HPV. Therefore, the E6/E7 oncoproteins are used for the construction of therapeutic vaccines against HPV-associated neoplasms. The purpose of this review is to discuss the results obtained with HPV16 E6/E7 oncoprotein based therapeutic vaccines in animal tumor models, as well as the prospects and limitations of the vaccines.     

Dietary intake and risk of persistent human papillomavirus (HPV) infection: the Ludwig-McGill HPV Natural History Study

The association between dietary intake and persistence of type-specific human papillomavirus (HPV) infection, during a 12-month period, among 433 women participating in the Ludwig-McGill HPV Natural History Study was evaluated by use of a nested case-control design. Dietary intake was assessed by a food-frequency questionnaire at the month-4 visit. HPV status was assessed at months 0, 4, 8, and 12 by polymerase chain reaction (MY09/11). Only women who ever tested positive for HPV were included in the present study: 248 had transient HPV infections (1 of 4 positive tests or nonconsecutively positive), and 185 had persistent HPV infections (> or =2 consecutive tests positive for the same HPV type). Risk of type-specific, persistent HPV infection was lower among women reporting intake values of beta-cryptoxanthin and lutein/zeaxanthin in the upper 2 quartiles and intake values of vitamin C in the upper quartile, compared with those reporting intake in the lowest quartile. Consumption of papaya > or =1 time/week was inversely associated with persistent HPV infection.     

Study of immortalization and malignant transformation of human embryonic esophageal epithelial cells induced by HPV18 E6E7

In order to study the effect of viruses and tumor promoters on the tumorigenicity of the esophagus, human embryonic esophageal epithelial cells were infected with human papilloma virus HPV18 E6E7-AAV in synergy with 12-O-tetradecanoylphorbol 13-acetate (TPA) to observe their malignant transformation. The cultured esophageal epithelial cells incubated with HPV18 E6E7-AAV were divided into two groups: the SHEEC1 group was exposed to TPA (5 ng/ml) for 4 weeks at the 5th passage of the cells; the SHEE group served as the control and was cultured in the same medium without TPA. The morphological phenotype, the DNA content during the cell cycle and the chromosomes were analyzed. The tumorigenicity was assessed by colony formation after cultivation in soft agar and transplanting the cells into nude mice. HPV18 E6E7 DNA was assayed by fluorescent in situ hybridization (FISH) and the polymerase chain reaction (PCR). The SHEE group, at its 20th passage, grew as a monolayer with the cells showing anchorage dependence and contact inhibition. The chromosome analysis showed diploidy, and soft-agar cultivation and injection into nude mice showed the cells to be non-tumorigenic. They were therefore immortalized cells. In contrast, the SHEEC1 group (TPA group) showed increased DNA synthesis and a proliferative index that was higher (45%) than that of the SHEE group (34%). The number of large colonies of dense multilayer cells (positively transformed foci) in soft agar was high in SHEEC1 group (4.0%) but low in the SHEE group (0.1%). Tumors resulting from transplantation were observed in all six nude mice injected subcutaneously with cells of the SHEEC1 group but no tumor developed in mice receiving cells of the SHEE group. In both groups of cells, HPV18 E6E7 DNA was positively detected by FISH and PCR. The malignant transformation of human embryonic epithelial cells was induced in vitro by HPV18 E6E7 in synergy with TPA. This is a good evidence for the close relationship between HPV and the etiology and pathogenicity of esophageal carcinoma. It is also a reliable model for studying the cellular and molecular mechanisms of carcinogenesis of esophageal carcinoma. Received: 1 October 1999 / Accepted: 2 February 2000     

Factors influencing the biological behaviour of cervical human papillomavirus (HPV) infections in prospectively followed-up women

To assess the natural history of Human papillomavirus (HPV) infections in uterine cervix, recently linked with cervical intraepithelial neoplasia (CIN), a long-term prospective follow-up study was started at our clinic in 1981. At this writing, a total of 418 women have been followed-up for a mean of 20 +/- 15 (M SD) months. On each attendance (at six-month intervals), the patient is subjected to colposcopy accompanied either by Papanicolaou (PAP) smears or punch biopsies. The latter are analysed for the cytopathic changes of HPV, for concomitant CIN, as well as for HPV structural proteins with IP-PAP technique. The local immunocompetent cell (ICC) infiltrates are analysed using ANAE technique to define B cells, MPS cells and T cells and monoclonal antibodies (McAb) for T cells subsets, NK (natural killer) cells and Langerhans cells. The relative levels of B- and T lymphocytes and MPS cells did not correlate with the clinical course, i.e. regression (RE), persistence (PE) or progression (PR) of the HPV lesions. The same was true with the expression of HPV antigens, intensity of the ICC infiltrate, as well as with the relative levels of NK (HNK-1+) cells and Langerhans (OKT-6+) cells. The T helper/T suppressor cell ratio was subject to minor fluctuations only as determined in three subsequent biopsies, and did not bear any meaningful relationship to the natural history of the HPV lesions. During the follow-up, 24.0% of the HPV lesions regressed, 54.9% remained persistent, and 21.1% progressed, 26 (6.2%) having been coned due to progression into CIS. The clinical course was most significantly influenced by the grade of HPV-associated CIN, to which RE was inversely and PR directly related. The results clearly confirm that cervical HPV infections are capable of progressing into CIS and thus show a natural history equivalent to that of classical CIN. Therefore, cervical HPV infections should be regarded as truly precancerous lesions, which should be examined, treated and followed-up with the same concern as the classical CIN.