Immunotherapeutic Effects of Dendritic Cells Pulsed with a Coden-optimized HPV 16 E6 and E7 Fusion Gene in Vivo and in Vitro

Background: : Cervical cancer is the second most common cause of cancer related death of women. PersistentHPV infection, especially with high-risk types such as HPV16 and HPV18, has been identified to be the primarycause of cervical cancer. E6 and E7 are the major oncoproteins of high-risk HPVs, which are expressed exclusivelyin HPV infected tissues, and thereby represent ideal therapeutic targets for immunotherapy of cervical cancer.Materials and Methods: In this work, we used recombinant adenovirus expressing coden-optimized HPV16 E6and E7 fusion protein (Ad-ofE6E7) to prime dendritic cells (DC-ofE6E7), to investigate the ability of primed DCvaccine in eliciting antitumor immunity in vitro and vivo. Results: Our results indicated that DC-ofE6E7 vaccineco-culturing with splenocytes could strongly induce a tumor-specific cytotoxic T lymphocyte (CTL) responseand kill the TC-1 cells effectively in vitro. Moreover, DC-ofE6E7 vaccine induced protective immunity againstthe challenge of TC-1 cancer cells in vivo. Conclusions: The results suggested that the HPV16 ofE6E7 primedDC vaccine has potential application for cervical cancer immunotherapy.     

A Novel Mutant of Human Papillomavirus Type 18 E6E7 Fusion Gene and its Transforming Activity

Background: Persistent human papillomavirus (HPV) infection, especially with high-risk types such as HPV16 and HPV18, has been identified as the primary cause of cervical cancer. E6 and E7 are the major onco-proteins of high-risk HPVs, which are consistently expressed in HPV infected tissues but absent in normal tissues and represent ideal therapeutic targets for immunotherapy of cervical cancer. Materials and Methods: In this study, the optimized fusion gene HPV18 E6E7 (HPV18 ofE6E7) was constructed according to genetic codon usage for human genes. At the same time, for safety future clinical application, a mutant of HPV18 ofE6E7 fusion gene was generated by site-directed mutagenesis at L52G for the E6 protein and C98G for the E7 protein. Results: HPV18-E6E7 mutant (HPV18 ofmE6E7) constructed in this work not only lost the transformation capability for NIH 3T3 cells and tumorigenicity in BALB/c nude mice, but also maintained very good stability and antigenicity. Conclusion: These results suggest that the mutant should undergo further study for application as a safe antigenspecific therapeutic vaccine for HPV18-associated tumors.     

人乳头瘤病毒16型E6/E7基因shRNA真核表达载体构建及其对人宫颈癌SiHa细胞增殖及迁移能力的影响

目的 构建针对人乳头瘤病毒16型E6/E7基因的shRNA真核表达载体,获得稳定表达干扰质粒的人宫颈癌SiHa细胞系并探讨其对SiHa细胞增殖及迁移能力的影响。方法 合成3条特异性干扰HPV16 E6/E7基因的shRNA片段并定向插入psilencer 2.1-U6 hygro载体,构建重组质粒psilencer 2.1-U6hygro-shE6/E7并转染入人宫颈癌SiHa细胞, Real- time PCR检测转染后细胞E6/E7 mRNA的表达,选择沉默效应最好的重组质粒并用潮霉素B稳筛,获得稳定表达重组质粒的SiHa细胞,并用real time-PCR和Western blot方法进行鉴定;分别运用CCK-8细胞增殖实验和细胞划痕愈合实验检测细胞的增殖及迁移能力。结果　测序证实针对HPV16 E6/E7的shRNA真核表达载体psilencer 2.1-U6 hygro-shE6/E7构建正确;转染psilencer 2.1-U6 hygro-shE6/E7的SiHa细胞HPV16 E6/E7表达明显受抑,同时其增殖及迁移能力也明显受抑制。结论 psilencer 2.1-U6 hygro-shE6/E7真核表达载体的成功构建并获得稳定沉默表达HPV16 E6/E7的人宫颈癌SiHa细胞系,证实沉默表达HPV16 E6/E7的SiHa细胞增殖及迁移能力会明显受到抑制,这为进一步研究HPV 16 E6/E7基因在宫颈癌发生发展过程中的功能奠定了实验基础。     

HPV16型E6、E7变异与宫颈癌发生发展的研究进展

宫颈癌是全球15 ～44岁女性中第二常见的恶性肿瘤,每年的死亡人数约为265 653人,在中国,宫颈癌的发生率及死亡率仍较高.高危型人乳头状瘤病毒(HPV)持续感染是宫颈癌前病变及宫颈癌发生的必要条件,HPV16是最常见的高危人乳头瘤病毒.HPV16编码的E6和E7蛋白在HPV相关的肿瘤中起关键作用.近年来的研究揭示了HPV16 E6、E7基因的变异引起氨基酸变化可影响E6、E7蛋白与p53、pRb 的结合,进而与宿主细胞恶性转化相关.本文将对近年来HPV16 E6、E7变异在宫颈癌发生发展中的作用作一综述.     

Human Papillomavirus Genotype Distribution and E6/ E7 Oncogene Expression in Turkish Women with Cervical Cytological Findings

Background: Infection with certain human papillomavirus (HPV) genotypes is the most important riskfactor related with cervical cancer. The objective of the present study was to investigate the prevalence of HPVinfection, the distribution of HPV genotypes and HPV E6/E7 oncogene mRNA expression in Turkish women withdifferent cervical cytological findings in Mersin province, Southern Turkey. Materials and Methods: A total of476 cytological samples belonging to women with normal and abnormal cervical Pap smears were enrolled in thestudy. For the detection and genotyping assay, a PCR/direct cycle sequencing approach was used. E6/E7 mRNAexpression of HPV-16, 18, 31, 33, and 45 was determined by type-specific real-time NASBA assay (NucliSENSEasyQ®HPV v1.1). Results: Of the 476 samples, 106 (22.3%) were found to be positive for HPV DNA by PCR.The presence of HPV was significantly more common (p<0.001) in HSIL (6/8, 75%) when compared with LSIL(6/14, 42.9%), ASC-US (22/74, 29.7%) and normal cytology (72/380, 18.9%). The most prevalent genotypes were,in descending order of frequency, HPV genotype 66 (22.6%), 16 (20.8%), 6 (14.2%), 31 (11.3%), 53 (5.7%), and83 (4.7%). HPV E6/E7 oncogene mRNA positivity (12/476, 2.5%) was lower than DNA positivity (38/476, 7.9%).Conclusions: Our data present a wide distribution of HPV genotypes in the analyzed population. HPV genotypes66, 16, 6, 31, 53 and 83 were the predominant types and most of them were potential carcinogenic types. Becauseof the differences between HPV E6/E7 mRNA and DNA positivity, further studies are required to test the roleof mRNA testing in the triage of women with abnormal cervical cytology or follow up of HPV DNA positive andcytology negative. These epidemiological data will be important to determine the future impact of vaccinationon HPV infected women in our region.     

Selective suppression of monocyte chemoattractant protein-1 expression by human papillomavirus E6 and E7 oncoproteins in human cervical epithelial and epidermal cells

Infection of cervical keratinocytes by high-risk HPV is involved in the etiology of cervical carcinoma. Since viral products are immunogenic, development of cancer may require suppression of immune responses directed against infected epithelial cells. Many markers of host immune effector responses decrease as cervical intraepithelial neoplasia progresses. Among these is epithelial cell expression of the chemokine MCP-1, though the mechanism for its suppression is unclear. Here, we show that the E6 and E7 viral oncogenes from high-risk HPV, individually and together, suppress MCP-1 expression in primary epithelial cells derived from the female genital tract. This is not a consequence of global suppression of chemokine expression since other chemokines, including IP-10, IL-8 and RANTES, were less affected. Furthermore, 4 of 6 HPV-positive cervical carcinoma cell lines did not express MCP-1. Our data indicate that suppression of MCP-1 expression is part of the program of high-risk HPV E6/E7-induced transformation of primary epithelial cells. These observations are consistent with a model in which MCP-1 expression by infected keratinocytes, which would stimulate an immune attack on HPV-transformed cells, is suppressed for invasive cervical cancer to appear.     

The synergistic therapeutic effect of cisplatin with Human papillomavirus E6/E7 short interfering RNA on cervical cancer cell lines in vitro and in vivo

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7-specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7-specific siRNA had greater efficacy than the combination of CDDP and E6-specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7-specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long-term exposure of HeLa cells to the combination of CDDP and E6/E7-specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7-specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7-specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.     

RNA干涉抑制宫颈癌 CaSki细胞株 HPV16 E6基因的研究

背景与目的：研究表明宫颈癌的发生发展与人乳头瘤病毒(human papilloma virus,HPV)E6、E7癌基因密切相关,于是人们采用核酶或HPV E6、E7反义寡核苷酸抑制其表达来治疗宫颈癌,虽然取得了一定的效果,但仍面临基因抑制效率低、维持时间短、工作量大、耗费多等问题。本研究采用最新的RNA干扰技术干扰宫颈癌CaSki细胞中HPV16 E6转录,以了解其能否特异性抑制HPV16 E6基因及其时效性如何。方法：设计合成针对HPV16 E6的荧光标记siRNA,借脂质体转染宫颈癌CaSki细胞,通过荧光照片计数荧光细胞占所有细胞的比例计算转染效率;测定转染后不同时间点的细胞凋亡率;RT-PCR测定HPV16 E6 mRNA变化,Western blot和流式细胞仪检测转染前后蛋白表达情况。结果：相差显微镜荧光照片显示细胞转染的效率为81％。HPV16 E6 siRNA转染细胞后24h、48h、5天凋亡率分别为7．7％、11．8％和37．4％,转染9天时凋亡率下降至12．6％。RT-PCR扩增结果显示,细胞转染前HPV16 E6 mRNA的量与siRNA阴性对照比较没有显著性差异,但转染后24h、48h、5天和9天分别减少了77％、83％、59％和41％;而作为内对照的β-actin mRNA在转染前后无变化。流式细胞仪定量测定HPV16 E6蛋白,结果显示转染后24h、48h和5天,蛋白表达抑制率分别为79．7％、80．4％和71．3％;9天时抑制率有下降,但仍可达57．4％。此结果与HPV16 E6 Western blot结果相吻合。以Lamin A/C作为内对照,不同的时间点Lamin A/C蛋白表达均无差异。结论：宫颈癌CaSki细胞中确实有RNA干扰现象存在,对外源性的HPV16病毒E6基因的干扰具有基因特异性和高效性。     

Diagnostic Performance of HPV E6/E7 mRNA and HPV DNA Assays for the Detection and Screening of Oncogenic Human Papillomavirus Infection among Woman with Cervical Lesions in China

Background: Human papillomavirus (HPV) is the most common sexually transmitted infection worldwide and it is responsible for most cases of cervical uterine cancer. Although HPV infections of the cervix do not always progress to cancer, 90% of cervical cancer cases have been found to be associated with high risk HPV (HRHPV) infection. HPV DNA testing is widely used, along with Papanicolaou (Pap) testing, to screen for cervical abnormalities. However, there are no data on the prevalence of genotype-specific HPV infections assessed by measuring HPV E6/E7 mRNA in women representative of the Chinese population across a broad age range. Materials and Methods: In the present study, we compared the results with the CervicGen HPV RT-qDx assay, which detects 16 HR-HPV genotypes (Alpha-9: HPV 16, 31, 33, 35, 52, and 58; Alpha-7: HPV 18, 39, 45, 51, 59, and 68; and Alpha-5, 6: HPV 53, 56, 66, and 69), and the REBA HPV-ID assay, which detects 32 HPV genotypes based on the reverse blot hybridization assay (REBA) for the detection of oncogenic HPV infection according to cytological diagnosis. We also investigated the prevalence and genotype distribution of HPV infection with a total of 324 liquid-based cytology samples collected in western Shandong province, East China. Results: The overall HPV prevalences determined by HPV DNA and HPV E6/E7 mRNA assays in this study were 79.9% (259/324) and 55.6% (180/324), respectively. Although the positivity of HPV E6/E7 mRNA expression was significantly lower than HPV DNA positivity, the HPV E6/E7 mRNA assay showed greater specificity than the HPV DNA assay (88.6% vs. 48.1%) in normal cytology samples. The prevalence of Alpha-9 (HPV 16, 31, 33, 35, 52, and 58) HPV infection among these women accounted for up to 80.3% and 76.1% of the high-grade lesions detected in the HPV mRNA and DNA tests, respectively. The HR-HPV genotype distribution, based on HPV DNA and E6/E7 mRNA expression by age group in patients with cytologically confirmed lesions, was highest in women aged 40 to 49 years (35.9% for cytologically confirmed cases, Pearson correlation r value=0.993, p<0.001) for high-grade lesions. Among the oncogenic HR-HPV genotypes for all age groups, there was little difference in the distribution of HPV genotypes between the HPV DNA (HPV -16, 53, 18, 58, and 33) and HPV E6/E7 mRNA (HPV -16, 53, 33, 58, and 18) assays. HPV 16 was the most common HPV genotype among women with highgrade lesions. Conclusions: Our results suggest that the HPV E6/E7 mRNA assay can be a sensitive and specific tool for the screening and investigation of cervical cancer. Furthermore, it may provide useful information regarding the necessity for early cervical cancer screenings and the development of additional effective HPV vaccines, such as one for HPV 53 and 58. Additionally, gaining knowledge of HPV distribution may also inform us about ecological changes in HPV after the vaccination.     

HPV16E7基因沉默对宫颈癌细胞系CaSki细胞增殖的影响

[目的]探讨脂质体转染HPVl6E7siRNA对人宫颈癌CaSki细胞增殖的影响。[方法]人工合成抑制HPVl6E7基因的siRNA片段,通过脂质体转染到CaSki细胞内,显微镜下观察其形态学变化;流式细胞术检测各组细胞周期变化;RT-PCR检测HPVl6E7mRNA的表达;Westernblot检测HPVl6E7蛋白表达。[结果]转染siRNA后的CaSki细胞,细胞增殖受到显著抑制．HPVl6E7mRNA表达显著下降,HPVl6E7蛋白水平显著下降。[结论]应用RNA干扰靶向抑制HPVl6E7基因可以显著抑制CaSki细胞增殖。     

宫颈癌及其癌前病变组织中HPV16 E6、HPV16 E7蛋白的表达及其意义

目的 探讨宫颈癌及其癌前病变组织中HPV16E6、E7蛋白的表达及其意义。方法 采用免疫组化SP法对15例正常宫颈、25例宫颈上皮内瘤变（C1N）以及61例浸润性宫颈癌组织进行了HPVl6E6、HPVl6E7蛋白表达的检测。结果在正常宫颈、CINI～Ⅱ、CINⅢ及浸润性宫颈癌中，HPVl6E6蛋白的阳性表达率分别为0（0／15）、7．14％（1／14）、36．36％（4／11）、59．02％（36／61）；CINⅢ和浸润性宫颈癌中HPVl6E6蛋白阳性表达率明显高于正常宫颈组织和CINI～Ⅱ（P〈0．05）；在高、中、低分化宫颈癌中，HPVl6E6蛋白阳性表达率分别为45．45％（5／11）、77．78％（14／18）、53．13％（17／32）；HPVl6E6蛋白在不同分化程度的宫颈癌组织中的阳性表达率有显著性差异（P〈0．05），但HPVl6E6蛋白表达与官颈癌组织分化程度无明显相关性（ys=0．123），HPVl6E6蛋白阳性表达率与宫颈癌临床分期和淋巴结转移无关（P〉0．05）。HPVl6E7蛋白在正常宫颈上皮、CINI-Ⅱ、CIN Ⅲ及浸润性富颈癌组织中的阳性表达率分别为20．00％（3／15）、42．86％（6／14）、63．64％（7／11）、57．38％（35／61），HPVl6E7蛋白在不同宫颈组织中的阳性表达率无明显差异（P=0．05）；HPVl6E7蛋白的表达与富颈癌临床分期、淋巴结转移和组织分化均无关（P〉0．05）。结论 HPVl6E6蛋白的检测有可能作为宫颈癌前病变转归的指标。     

Immortalization without neoplastic transformation of human mesenchymal stem cells by transduction with HPV16 E6/E7 genes

hMSCs derived from bone marrow are useful as a species-specific cell culture system for studying cell lineage differentiation and tissue remodeling. However, hMSCs usually have a short in vitro life span due to replicative senescence. We therefore used a high dose of retroviral vector LXSN-16E6E7 to transduce hMSCs of an aging donor and obtained an actively proliferating cell line, designated KP-hMSCs, which expressed HPV16 E6/E7 mRNA. Whereas parental hMSCs ceased to grow after 30 PDs, KP-hMSCs could be propagated beyond 100 PDs. With culture procedures to avoid selection pressure and crowded cell growth, KP-hMSCs showed no signs of neoplastic transformation as examined by soft-agar anchorage-independent growth and NOD-SCID mouse tumorigenicity assays. KP-hMSCs gave similar cytofluorimetric profiles of 31 CD markers to those of the parental primary hMSCs, except with some morphologic changes and expansion of an originally very minor CD34(dim)CD38(+)CD50+ cell population. Upon exposure to specific stimulating conditions in vitro, KP-hMSCs could respond and differentiate along the mesenchymal (bone, fat and cartilage) and nonmesenchymal (neuron) cell lineages. Our results indicated that hMSCs could be immortalized by transduction with HPV16 E6/E7, maintained without neoplastic transformation by careful culture procedures and thus useful for stem cell research and clinical application.     

HPV16Z-Hsp65-E6／E7无佐剂重组蛋白疫苗的研究

目的：研究治疗性HPV16Z-Hsp65-E6／E7无佐剂重组蛋白疫苗的抗肿瘤活性。方法：通过淋巴细胞增殖实验和细胞毒性杀伤实验研究该疫苗激发的细胞免疫反应及反应强度；观察该疫苗对小鼠TC-1肿瘤细胞移植瘤的体内治疗作用和对小鼠生存期的影响。结果：重组蛋白疫苗免疫小鼠后，小鼠脾淋巴细胞与该疫苗体外混合培养增殖明显，并可特异性地在体外杀伤TC-1细胞；体内抑瘤试验显示该疫苗对HPV16病毒转化的TC-1细胞小鼠移植瘤的生长有显著的抑制作用。结论：该疫苗能激发特异性细胞免疫反应；显著抑制HPV16转化的TC-1肿瘤细胞生长。     

Human Papillomavirus Type 16/18 Oncoproteins: Potential Therapeutic Targets in Non-smoking Associated Lung Cancer

High-risk human papillomavirus (HPV) especially HPV-16 and HPV-18 types are speculated to be importantrisk factors in non-smoking associated lung cancer in Asia. Increasing evidence has demonstrated that HPVoncoproteins may contribute to lung tumorigenesis and cell transformation. Importantly, HPV 16/18 E6 and E7oncoproteins can mediate expression of multiple target genes and proteins, such as p53/pRb, VEGF, HIF-1α,cIAP-2, and hTERT, and contribute to cell proliferation, angiogenesis and cell immortalization through differentsignaling pathways in lung cancer. This article provides an overview of experiment data on HPV-associated lungcancer, describes the main targets on which HPV E6/E7 oncoproteins act, and further discusses the potentialsignaling pathways in which HPV E6/E7 oncoproteins are involved. In addition, we also raise questions regardingexisting problems with the study of HPV-associated lung cancer.     

HPV18 E6E7 反义荧光真核表达载体的构建及其在宫颈癌HeLa 细胞中的表达

 目的 构建HPV18型E6E7反义荧光真核表达载体,并观察其对宫颈癌HeLa细胞中HPV18 E6和E7基因表达的影响,探索反义技术用于治疗临床HPV感染及宫颈癌的可能性。方法 以HPV18型全基因质粒为模板,PCR法扩增HPV18型E6E7区716bp片段,利用基因重组技术将目的片段反向插入荧光真核表达载体pEGFP-C1,EcoR I酶切并测序鉴定;采用脂转法将重组质粒pEGFP-HPV18 E6E7as（EGFP-18AS）转染宫颈癌HeLa细胞株,通过RT-PCR及western blot检测细胞中E6、E7 mRNA和蛋白的表达。结果 成功构建HPV18E6E7反义荧光真核表达载体EGFP-18AS,经脂质体转染HeLa细胞,48h后在荧光倒置显微镜下可见明显的绿色荧光,且细胞中E6、E7 mRNA及蛋白表达水平均明显下调。结论 反义荧光真核表达载体可以有效的抑制HPV18E6、E7癌基因的表达,为治疗HPV感染和宫颈癌提供了一种新的方法。     

Eight-type human papillomavirus E6/E7 oncoprotein detection as a novel and promising triage strategy for managing HPV-positive women

The management of HPV-positive women becomes particularly crucial in cervical cancer screening. Here we assessed whether detection of E6 or E7 oncoproteins targeting eight most prevalent HPV types could serve as a promising triage option. Women (N = 1,416) aged 50–60 from Shanxi, China underwent screening with HPV testing and liquid-based cytology (LBC), with any positive results referring to colposcopy and biopsy if necessary. Women with HPV-positive results received further tests using DNA-based genotyping, E6 or E7 oncoprotein detection targeting HPV16/18 (for short: E6 (16/18) Test) or HPV16/18/31/33/35/45/52/58 (for short: E6/E7 (8 types) Test), respectively. Among HPV-positive women, E6/E7 (8 types) oncoproteins had lower positivity (17.37%) compared to DNA-based genotyping for same eight types (58.30%) and LBC with ASC-US threshold (50.97%); HPV16 was the genotype showing the highest frequency (8.49%) for oncoprotein detection followed by HPV52 (3.47%), 58 (2.32%), 33 (1.54%), 18 (1.16%), 45 (0.77%), 35 (0.39%) and 31 (0%). For detection of cervical intraepithelial neoplasia Grade 3 or higher (CIN3+), E6/E7 (8 types) Test had similar sensitivity (100.00%) and superior specificity (85.94%) as well as positive predictive value (PPV, 22.22%) compared to both LBC and DNA-based genotyping (8 types); For detection of CIN2+, E6/E7 (8 types) Test was less sensitive (67.74%) but still more specific (89.47%) and risk predictive with PPV of 46.67%. Notably, E6/E7 (8 types) Test remarkably decreased the number of colposcopies needed to detect one CIN2+ and CIN3+ (2.14 and 4.50). E6/E7 oncoprotein detection showed a good “trade-off” between sensitivity and specificity with more efficient colposcopy referrals, which is of great importance to maximize the benefits of HPV-based screening program, especially applicable for the areas with high HPV prevalence and low-resources.     

HPV E6/E7 mRNA和HPV DNA检测技术在宫颈上皮内瘤变中的诊断价值分析

目的比较人乳头瘤病毒(HPV)E6/E7 mRNA和HPV DNA检测技术对宫颈上皮内瘤变(CIN)2级及以上(CIN2+)患者的诊断价值,并评价HPV E6/E7 mRNA检测结果在不同实验室间的一致性。方法采用HPV E6/E7 mRNA和HPV DNA检测技术对212例门诊体检的健康者和住院的宫颈病变患者的宫颈脱落细胞学标本进行检测。以病理诊断结果为金标准,评价两种检测技术诊断CIN2+的灵敏度和特异度。北京市迪安中心实验室和北京市怀柔妇幼保健院实验室均采用HPV E6/E7 m RNA检测技术检测同一批标本,评价实验室间检测的一致性。结果HPV E6/E7 m RNA检测的阳性率为38.7%,与HPV DNA的阳性率43.9%比较,差异无统计学意义(P﹥0.05)。HPV E6/E7 mRNA和HPV DNA的检测阳性率均随着病理分级的升高而增加(P<0.01)。HPV E6/E7mRNA检测CIN2+的灵敏度为92.96%,与HPV DNA的90.14%相比,差异无统计学意义(P﹥0.05),而HPV E6/E7mRNA检测CIN2+的特异度为88.65%,高于HPV DNA的79.43%,差异有统计学意义(P<0.05)。两个实验室采用HPV E6/E7 m RNA检测阳性一致的标本例数为78,阴性一致的标本例数为121,总一致率为93.87%,Kappa=0.872,一致性较好。结论与HPV DNA检测技术相比,HPV E6/E7 mRNA检测宫颈病变的特异度更具优势,实验室间重复性检测的一致率较高,有望成为中国宫颈癌HPV筛查的首选方法。     

液基细胞学联合 HPV mRNA 检测对宫颈癌的诊断价值

目的：探索高危型 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 两种检测方法分别联合宫颈薄层液基细胞学检查（TCT）应用于宫颈癌早期诊断的临床意义。方法：选择2013年1月至2014年12月我院妇科门诊就诊行 TCT 检查的259例检测标本,对高危型 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 进行检测,结合 xb 细胞病理学分级和组织病理学诊断进行统计分析。结果：纳入研究的259例患者中 HPV E6／E7 mRNA 检测阳性率为35．1％（91／259）,HPV E6／E7 DNA 检测阳性率为52．1％（135／259）。NILM、ASCUS、LSIL、HSIL 四种细胞病理学分级 HPV E6／E7 mRNA 和 HPV E6／E7 DNA 检出率分别为23．5％、29．4％、64．3％、66．7％和32．4％、52．5％、61．9％、83．3％。对于正常组织,HPV E6／E7 mRNA 的敏感度、准确度和阳性预测值均低于HPV E6／E7 DNA（P ＜0．05）,但特异度和阴性预测值高于 HPV E6／E7 DNA（P ＜0．001）。对于 CIN1组织, HPV E6／E7 mRNA 的敏感度、特异度和阳性预测值均低于 HPV E6／E7 DNA（P ＜0．05）,但准确度和阴性预测值高于 HPV E6／E7 DNA（P ＜0．001）。在 CIN2和 CIN3组织中,HPV E6／E7 mRNA 的准确度和阴性预测值均高于 HPV E6／E7 DNA（P ＜0．001）,HPV E6／E7 mRNA 的敏感度、特异度、阳性预测值与 HPV E6／E7 DNA 无统计学差异（P ＞0．05）。结论：高危型 HPV E6／E7 mRNA 检测能更准确地反映病毒感染后的活化状态,HPV宫颈薄层液基细胞学检查联合高危型 HPV E6／E7 mRNA 检测可提高宫颈癌早期筛查的准确性。     

HPV16 E6 E7 siRNA作用于人宫颈癌细胞株的实验研究

目的: 使用HPV16 E6、E7 siRNA处理表达HPV16阳性的人宫颈鳞癌细胞株CaSki和SiHa,观察其对HPV16 E6、E7原癌基因的特异性抑制作用,为探讨使用siRNA治疗宫颈癌的可行性提供实验基础. 方法: 分别设计并合成靶向于HPV16 E6、E7的siRNA各3条,经脂质体包裹后转染两株细胞,分别采用RT-PCR和Western Blot方法,通过对转染前后各时间段两基因mRNA及蛋白的表达情况的检测,验证RNAi作用的特异性及时效性. 结果: 同对照组相比,各E6、E7 siRNA均可引起靶基因表达水平的显著性下降(P<0.05);而空脂质体组及阴性对照siRNA组的靶基因表达水平则无明显变化.其中E6-1 siRNA和E7-2 siRNA对靶基因抑制作用较强.E6-1 siRNA和E7-2 siRNA转染两株细胞后24h、48h、72h及96h均可观察到靶基因表达的显著性降低(P<0.05),而对相应的非靶基因无明显抑制作用. 结论: 不同序列的E6、E7 siRNA,对靶基因的干扰效率不同;E6、E7 siRNA分别作用于宫颈鳞癌细胞株,均引起了靶基因的特异、高效性的抑制,而时非靶基因的表达无明显变化,且抑制作用至少维持到转染后96h.为进一步研究采用RAN干扰技术进行宫颈癌的生物治疗奠定了基础.