宫颈癌及癌前病变中p16INK4A与HPV的关系

高危型人乳头瘤状病毒(HR-HPV)持续感染宫颈后,通过其基因产物干扰正常的细胞周期调节,导致细胞的连续增殖与恶性转化.p16INK4A基因产物是细胞周期素依赖性激酶抑制物(CDKI),负性调节细胞周期,由于HPV E7蛋白阻断了p16INK4A的细胞周期调节级联,因此在HR-HPV感染的宫颈癌及癌前病变组织中出现p16INK4A过表达.p16INK4A与HPV联合监测将提高宫颈癌筛查的有效性,有助于宫颈癌的诊断与鉴别诊断.     

HPV16 E5蛋白在宫颈癌发生中的作用

人乳头瘤病毒16型（HPV16）与宫颈癌密切相关,编码E5蛋白。HPV16 E5蛋白是一种细胞膜整合蛋白,主要分布于高尔基体和内质网。大量研究证明,HPV16 E5蛋白通过影响表皮生长因子受体信号转导途径及环氧化酶2（COX-2）途径的活性,增加宿主细胞的免疫逃避,调节细胞周期,促进细胞转化、增殖,减少细胞凋亡,促进肿瘤中新生血管的形成等机制影响宫颈癌的发生与发展。就HPV16 E5蛋白的分子生物学特点及与宫颈癌细胞增殖侵袭凋亡关系的研究进展进行综述。     

人乳头瘤病毒E5的研究进展

宫颈癌是女性常见的恶性肿瘤之一,宫颈疾病与人乳头瘤病毒相关。人乳头瘤病毒E5、E6、E7是重要的致癌蛋白,E5多在疾病早期表达,深入探讨人乳头瘤病毒E5的致病机制将为治疗相关宫颈疾病提供契机,并为开发治疗性宫颈癌疫苗提供新思路。     

人乳头瘤病毒E2蛋白结构和功能研究进展

高危型人乳头瘤病毒(HPV)是导致宫颈癌的主要原因。HPV E2蛋白在病毒的转录和复制调节中起着重要的作用。其主要由氨基端转录活化结构域、羧基端DNA结合域以及可变的铰链区组成。HPV E2基因的突变或整合和致癌机制有关。HPV E2蛋白能抑制E6和E7蛋白表达,HPV整合常在E2基因处发生断裂缺失,而E2基因的缺失导致E6和E7蛋白的过表达。就有关HPV E2蛋白的结构和功能的研究进展综述,为HPV E2蛋白在致癌机制中作用的研究提供理论基础。     

腺病毒伴随病毒表达载体表达人乳头状瘤病毒16型E6/E7基因的构建及应用

目的　为了了解人乳头状瘤病毒 (Humanpapillomavirus ,HPV) 1 6型的E6 /E7基因在细胞恶性转化中所起的作用 ,利用腺病毒伴随病毒载体 (AAVHelper -FreeSystem)构建和表达人乳头状瘤病毒 1 6型E6 /E7基因。方法　在pLXSN1 6E6E7质粒中经PCR扩增回收HPV 1 6E6E7基因片段 ,连接于T载体上进行测序 ,将正确的HPV 1 6E6E7插入pAAV -IRES -hrGFP质粒 ,协同pAAV -RC质粒和pHelper质粒共转染HEK 2 93细胞 ,包装表达HPV 1 6E6E7基因的重组腺病毒伴随病毒 ,收获病毒 ,并检测病毒的感染效率。结果　在包装细胞系HEK 2 93细胞中能形成较高感染效率的腺病毒伴随病毒 ,激光共聚焦检测可发现HEK 2 93细胞内有绿色荧光蛋白表达 ,HEK 2 93细胞经PCR可扩增出特异性的HPV 1 6E6E7基因片段 ,经流式细胞仪检测重组病毒的感染效率为71 3%。结论　携带人乳头状瘤病毒 1 6型E6E7基因的腺病毒伴随病毒可感染细胞 ,并在细胞内表达 ,可望用于宫颈癌病因学的研究     

人乳头状瘤病毒E6、E7原癌蛋白致癌的机制

高危型人乳头状瘤病毒E6、E7基因编码的原癌蛋白是导致宫颈癌的重要原因.其致癌机制主要与p53、pRb有关,还与端粒酶激活、干扰素调控因子3、E6靶蛋白1、p21、组蛋白脱乙酰脂酶以及凋亡有关.     

FHIT基因、HPV16E6的表达与宫颈鳞癌发病的相关性研究

目的:探讨抑癌基因脆性组氨酸三联(fragile histine triad,FHIT)基因、人乳头瘤病毒16E6(HPV16E6)蛋白在宫颈鳞癌中的表达及其相互关系.方法:应用免疫组织化学链霉菌抗生物素蛋白-过氧化物酶连接(SP)法观察40例宫颈鳞6,30例宫颈上皮内瘤样病变(CIN)和30例正常宫颈组织中MIT基因...     

高危型人乳头瘤病毒E6/E7 mRNA与其DNA检测在子宫颈病变中的对比研究

目的探讨高危型人乳头瘤病毒(HR-HPV)E6/E7 m RNA及HPV DNA检测与宫颈病变的关系。方法选取2013年1月至2015年12月在青岛市市立医院妇科门诊行宫颈癌联合筛查的两组女性:LCT+HRHPV DNA(HC2组,n=2 417);LCT+HR-HPV E6/E7 m RNA(E6/E7组,n=2 906)。细胞学ASCUS+或HPV阳性转诊阴道镜行宫颈活检。计算HPV m RNA E6/E7与HPV DNA在不同病理结果中的阳性率、病毒载量及在CIN2+中的一致率。结果 (1)在病理正常、CIN1、CIN2、CIN3和ICC中E6/E7 m RNA阳性率分别为61.36%、76.00%、92.11%、96.00%和100.00%,随病变程度加重而升高(P0.05)。正常病理结果中E6/E7组HPV的阳性率低于HC2组(χ2=7.764,P=0.005)。(2)E6/E7组病毒载量的中位数和四分位数在不同病理结果中分别为629.72(339.31,2 122.94),1117.33(319.17,3 101.88),1 537.12(422.19,4 219.05),3 396.59(1 217.32,6 316.81),10 860.43(3 473.19,29 662.29),随着病变程度加重逐渐升高,差异有统计学意义(P0.05)。(3)在CIN2+患者中,E6/E7组和HC2组的一致率为82.64%,Kappa值=0.69。结论 HPV E6/E7阳性率与HC2一样随着宫颈病变程度的加重逐渐升高。HPVE6/E7 m RNA与HPV DNA诊断CIN2+的一致性较好。HPV E6/E7载量可能反映病变程度,但还需进一步临床验证。     

宫颈上皮内瘤变及早期宫颈癌组织中P16、HPV L-1壳蛋白的表达及与HR-HPV载量相关性研究

目的:探讨宫颈上皮内瘤变(CIN)及早期宫颈癌组织中P16、HPV L-1壳蛋白的表达及与高危型人乳头瘤病毒(HR-HPV)载量的相关性。方法:分别采用免疫组化法和第二代杂交捕获法(hybrid captureⅡ,HC-2)检测26例慢性宫颈炎组织、83例低度鳞状上皮内病变(LSIL)(CINⅠ83例)、109例高度鳞状上皮内病变(HSIL)(CINⅡ49例,CINⅢ60例)、11例早期宫颈鳞癌组织中P16蛋白、HPV L-1壳蛋白的表达及HR-HPV载量,并分析其相关性。结果:1在宫颈癌前病变中随病变级别增高,HR-HPV阳性率增加,差异有统计学意义(P0.01)。各组织学分级中病毒载量分布差异有统计学意义,HSIL(44.95%)及早期宫颈癌组织(63.64%)中皆以低病毒载量(1~100 RLU/CO)为主。从慢性宫颈炎、LSIL、HSIL到早期宫颈癌,P16蛋白阳性表达率分别为11.54%(3/26),55.42%(46/83),85.32%(93/109),100.00%(11/11),差异有统计学意义(P0.01);L-1壳蛋白阳性率分别为15.38%(4/26),28.92%(24/83),14.68%(16/109),0.00%(0/11),差异有统计学意义(P0.05)。2229例宫颈组织中,随HPV载量增加,P16蛋白表达增强;L-1壳蛋白阳性表达率增加,差异有统计学意义(P0.01)。3在慢性宫颈炎组中,P16蛋白阳性表达与HPV载量呈正相关(r=0.491,P0.05)。在LSIL组中,P16蛋白与HPV载量(r=0.459,P0.01)及L1壳蛋白表达(r=0.297,P0.01)皆呈正相关。4在HSIL及早期宫颈癌组中,P16蛋白、L-1壳蛋白表达与HPV载量三者之间无明显相关性(P0.05)。结论:P16、HPV L-1壳蛋白异常表达是宫颈癌前病变发生发展的早期分子事件,对判断CINⅠ有参考价值,可能比HR-HPV载量更具有预测价值。     

人乳头瘤病毒疫苗预防宫颈癌的应用

宫颈癌严重危害女性健康,现已明确人乳头瘤病毒(HPV)感染是其主要致病因素。HPV通过机体的细微损伤入侵,HPV E6和E7癌蛋白中的1种或2种持续表达是高危型HPV感染致瘤的关键所在,检测高危型HPV感染及病毒癌蛋白仍不能有效预防宫颈癌。研究者们正着手研制针对HPV的病毒疫苗,从源头预防HPV感染,以期实现宫颈癌的一级预防。目前已有针对HPV16/18型的二价疫苗Cervarix和针对HPV16/18/11/6型的四价疫苗Gardasil的认证上市,预防性HPV疫苗已在全球范围内推广使用并取得显著效果。新一代预防性HPV疫苗在解决疫苗的成本、持久性和广谱免疫问题上取得突破性进展,宫颈癌有望成为人类抗肿瘤史上第一个可以预防的癌症。综述近年HPV的生物学特性、致病机制及预防性HPV疫苗的应用研究与现状。     

HPV单一感染与多重感染对宫颈病变影响的比较

目的：探讨单一高危HPV亚型与多重高危HPV亚型感染对宫颈病变的影响。方法：选取2010年1月—12月在天津医科大学总医院妇科门诊就诊的有性生活史并出现临床症状的患者137例,比较患者细胞学、病理学结果出现的概率;比较单一高危HPV亚型与多重高危HPV亚型感染对宫颈病变影响的差别。结果：①在全部感染病例中,以20～49岁者居多,并且随着年龄的增长,感染率呈现下降趋势。②感染病例中,高危型别以HPV16感染率最高。③在宫颈高级别病变中,以HPV16感染率最高。④单一高危与多重高危HPV感染相比较,宫颈细胞学和病理学结果差异无统计学意义。⑤高低危混合HPV感染中低级别病变概率高于单纯高危HPV感染,而高级别病变则相反。结论：①与单一高危HPV亚型相比,多重高危HPV亚型感染对宫颈病变严重程度的影响不明显。②与单纯高危HPV亚型感染相比,高低危HPV亚型混合感染更多表现为低级别宫颈病变。     

人乳头瘤病毒检测及分型基因芯片制备的初步研究

目的开发研制对人乳头瘤病毒(HPV)进行检测及分型的基因芯片。方法2003年6月至2003年12月郑州大学第二附属医院将人工合成的HPV通用探针,HPV6,11,16,18特异性寡核苷酸片段作为实验组探针,将与HPV没有同源性的植物基因片段作为内参照基因点样于玻片介质,制成HPV检测及分型芯片,将HPV标准品荧光标记后与芯片杂交,用GMS418扫描仪对芯片进行扫描,以HPV标准品与芯片的杂交结果对芯片检测方法的准确性进行评价。结果该芯片的内参照结果能做到绝对阳性和绝对阴性,各HPV标准品的芯片检测结果显示其在芯片上的相应位置为明显的阳性信号,且各型别之间无错配杂交信号。结论本研究制作的HPV检测及分型芯片可以成功用于HPV的检测及分型,可望用于临床。     

A pilot study to investigate the treatment of cervical human papillomavirus infection with zinc-citrate compound (CIZAR®)

Objective

In the present study the potential therapeutic effects of zinc-citrate compound (CIZAR®) in women infected with high-risk human papillomavirus (HR-HPV) was investigated.

Methods

A total of 194 women diagnosed with HR-HPV infection using the Hybrid capture (HC) II assay with no evidence of high grade squamous intraepithelial lesions (HSIL) or worse by Pap smear and colposcopy were enrolled. Among them, 76 women were treated by twice weekly self administered intra-vaginal infusion of 0.5 mM zinc citrate solution containing CIZAR® for 12 weeks and were evaluated for clearance of the HR-HPV infection compared to 118 women without treatment (Control group).

Results

The 12 weeks zinc citrate solution treatment resulted in the elimination of HR-HPV in 49/76 (64.47%) patients compared to the spontaneous clearance of 15.25% (18/118) in the control group (p = 0.000). By logistic regression analysis, the 12 week zinc citrate solution treatment reduced the risk of persistent HR-HPV infection significantly (OR 0.079; 95% CI 0.039-0.165; p = 0.000).

Conclusion

The results of this study showed for the first time that treatment with intra-vaginal infusion of a zinc-citrate compound (CIZAR®) can result in elimination of HR-HPV infection from the uterine cervix.     

Methylation of HPV16 genome CpG sites is associated with cervix precancer and cancer

Objective.

Invasive cervix cancer (ICC) is the second most common malignant tumor in women. Human papillomavirus 16 (HPV16) causes more than 50% of all ICC and is a major cause of cervix intraepithelial neoplasia (CIN). DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides. Such epigenetic modifications are associated with changes in DNA-protein interactions and gene activation. This study examined the association of viral and host genomic methylation patterns and cervix neoplasia.

Methods.

Exfoliated cervical lavage samples positive for HPV16 from women with and without cytomorphic changes of infection (n = 46), CIN2 (n = 12), and CIN3+ (n = 27) were used to interrogate the methylation patterns of the HPV16 L1 gene and upstream regulatory region (URR), five host nuclear genes (TERT, RARB, DAPK1, MAL, and CADM1), and mitochondrial DNA (mtDNA). DNA isolated from exfoliated cervicovaginal cells was treated with bisulfite, specific regions of the viral and host genome were PCR amplified and CpG methylation was quantified using EpiTYPER and pyrosequencing.

Results.

Methylation at 14 of the tested CpG sites within the HPV16 L1 region were significantly higher in CIN3+ compared to HPV16 genomes from women without CIN3+. In contrast, 2/16 CpG sites in HPV16 URR, 5/5 in TERT, 1/4 in DAPK1 and 1/3 mtDNA, and 2/5 in RARB were associated with increased methylation in CIN3+.

Conclusions.

These results indicate that increased methylation of CpG sites in the HPV16 L1 ORF is associated with CIN3+ and, thus, may constitute a potential biomarker for precancerous and cancerous cervix disease.     

hrHPV E6/E7 mRNA用于宫颈癌机会性筛查的临床价值

目的:研究hrHPVE6/E7 mRNA在宫颈筛查中的准确性,探索其用于宫颈癌机会性筛查的临床价值。方法:选取2013年1月至2015年12月在青岛市市立医院及城阳区人民医院妇科门诊行机会性筛查的女性共7791例,年龄25~65岁。所有受试女性均行宫颈液基薄层细胞学检查(LBC)。每位受试者同时行HPV检测,根据采取的HPV方法不同分为3组:HC2组(2417例),HPV分型组(2456例),E6/E7组(2906例)。比较4种筛查方法的灵敏度、特异度、阳性预测值、阴性预测值、阴道镜转诊率、CINⅡ+/CINⅢ+检出率。结果:E6/E7检测CINⅡ+/CINⅢ+的灵敏度分别为93.02%和94.83%,NPV分别为99.52%和99.88%,与HC2及HPV分型之间无统计学差异(P0.05)。E6/E7检测CINⅡ+/CINⅢ+的特异度分别为90.20%和86.90%,与LBC、HC2及HPV分型两两比较均有统计学差异(P0.01)。以HC2和HPV分型为参照时,E6/E7检测CINⅡ+/CINⅢ+的RR均1。E6/E7的阴道镜转诊率为14.73%,低于另两种HPV检测方法,高于LBC,差异有统计学意义(P0.01)。3种HPV检测方法的CINⅡ+/CINⅢ+检出率无统计学差异(P0.05)。结论:hrHPV E6/E7 mRNA与HC2和HPV分型相比,灵敏度相同、特异度高,阴道镜转诊率低,CINⅡ+/CINⅢ+检出率无差别,可用于宫颈癌的机会性筛查。     

A phase II trial of interleukin-12 in patients with advanced cervical cancer: clinical and immunologic correlates. Eastern Cooperative Oncology Group study E1E96

PURPOSE: The ability to mount lymphoproliferative responses to peptides derived from the human papillomavirus (HPV) E6 and E7 oncoproteins has been associated with regression of dysplastic lesions of the uterine cervix and loss of associated HPV infection. Interleukin-12 (IL-12) is a potent immunopotentiator of T-cell function, and has been shown in phase I clinical trials to be tolerable. EXPERIMENTAL DESIGN: Patients were required to have measurable metastatic, recurrent or inoperable cervical carcinoma. Patients could have had one prior adjuvant regimen and one prior regimen for advanced disease. Treatment consisted of IL-12 administered at 250 ng/kg IV as a rapid push in the outpatient setting daily x 5 every 21 days. Whole blood samples were acquired twice before treatment then approximately every 3 weeks to assess lymphoproliferative response in vitro to HPV type 16 (HPV 16) E4, E6, and E7 peptides. These responses were correlated with demographics and with clinical outcome. RESULTS: Thirty-four patients were enrolled; 29 were evaluable. Over half had received cisplatin-based chemotherapy. The most common serious toxicities were hematologic or hepatic, and all were reversible. There was one partial responder (3%). The median survival was 6.5 months (95% CI: 5.8, 11.5 months). Eighteen of 29 eligible patients had evaluable laboratory data both pre- and post-therapy. There was a statistically significant increase in lymphoproliferative responses for HPV 16 E4, E6, and E7 peptides (P=0.020, 0.020, 0.043). There was a significant association between change in lymphoproliferative response to HPV 16 E6 peptides and number of cycles of treatment administered (P=0.048). There was no correlation between change in lymphoproliferative response to any peptide with age, performance status, race, prior chemotherapy, time from diagnosis to treatment, or with overall survival. CONCLUSIONS: IL-12 treatment was associated with improved lymphoproliferative responses to HPV 16 E4, E6, and E7 peptides. This is the first clinical trial to demonstrate induction of cell-mediated immune (CMI) responses to specific antigens (peptides) following treatment with IL-12 in women with cervical cancer. This improvement in immune response was not associated with enhanced objective response or survival.     

Influence of Sources of Information and Parental Attitudes on Human Papillomavirus Vaccine Uptake among Adolescents

Study ObjectiveThe purpose of this study was to: 1) describe parental sources of information about human papillomavirus (HPV) vaccination for adolescents, 2) understand how parental sources of information about HPV vaccine are associated with adolescent HPV vaccine uptake, and 3) understand if the relationship between a greater number of HPV-related information sources and HPV vaccine uptake among adolescents is mediated by parental attitudes.Design, Setting, Participants, and InterventionsWe conducted a 3-arm randomized controlled trial in middle and high schools in eastern Georgia from 2011 to 2013. As part of the trial, we surveyed parents during the final year to understand their sources of information about HPV vaccine for their adolescent. Data were collected from 360 parents via phone and online surveys.Main Outcome MeasuresParents responded to a survey that asked them to identify demographic information, parental HPV attitudes, sources of information about HPV vaccination, and HPV vaccine uptake.ResultsMost of the sample was African American (74%; n = 267) and 53% of parents (n = 192) reported that their adolescent received at least 1 HPV vaccine dose. The top sources of information about HPV vaccine reported by parents were a doctor or medical professional (80%; n = 287) and television (64%; n = 232). A mediation analysis showed sources of information about HPV vaccine are associated with parental attitudes, and parental attitudes about HPV vaccine are associated with vaccine uptake among adolescents.ConclusionThese findings highlight the importance of HPV sources of information on parental attitudes.     

Short-term type-specific HPV persistence and its predictors in an asymptomatic general female population in Zhejiang, China

Objective

To study short-term type-specific human papillomavirus (HPV) persistence and associated predictors in an asymptomatic general female population in Zhejiang, China.

Methods

Persistence was evaluated in women who were HPV positive at enrollment and who returned within 6–14 months. Liquid-based cytology screening was encouraged for returning women.

Results

Persistence was evaluated in 548 women. Overall persistence was 49.1%, and established high-risk HPV persistence was 54.5%. The types associated with the highest level of persistence were HPV-52, HPV-58, HPV-56, and HPV-16—most of which belong to α9 species. In total, 252 women refused cytology screening. No differences were found regarding HPV persistence and other variables among women who returned for follow-up, women who accepted cytology screening, and women who refused cytology screening. Among women aged 35 years or older, there were no differences between those with normal cytology and those with abnormal cytology in the short-term persistence of HPV types, except for HPV-58 (P < 0.01) and HPV-81 (P = 0.04). Established high-risk HPV persistence increased with age, low income, and early sexual experience.

Conclusion

The data support close surveillance of older women with established high-risk HPV infections, and conservative management of women with non-α9 HPV and no risk factors.     

宫颈癌患者外周血HPV16抗原特异性CD8~+细胞毒性T细胞的检测及意义

目的:初步探讨人乳头瘤病毒16型(HPV16)阳性宫颈癌患者细胞免疫状况,了解其外周血抗原特异性CD8~+细胞毒性T细胞的水平.方法:采取免疫荧光标染重组MHC Ⅰ类分子-肽五聚体技术,运用流式细胞术定量检测患者和正常人外周血CD8~+细胞毒性T细胞、记忆性细胞毒性T细胞、活化细胞毒性T细胞及HPV16抗原特异性CD8~+细胞毒性T细胞.结果:宫颈癌患者CD8~+细胞毒性T细胞计数、记忆性细胞毒性T细胞计数、体内抗原特异性CD8~+细胞毒性T细胞计数和对照组比较差异无统计学意义,但活化细胞毒性T细胞计数低于对照组(P=0.322),体外经抗原肽刺激后抗原特异性CD8~+细胞毒性T细胞计数高于对照组(P=0.0068).结论:HPV16 阳性宫颈癌患者外周血中活化细胞毒性T细胞数目减少,表明患者细胞免疫功能受到一定的影响,通过抗原表位有效的诱导是提高患者的特异性细胞免疫功能的重要途径,运用五聚体技术检测宫颈癌患者抗原特异性细胞毒性T细胞,可初步反映患者特异性细胞免疫状态并有利于进行复发和预后分析.