HPV16 E6E7抗原表达模型的建立

目的 用基因重组技术构建pcDNA-E6E7真核表达载体.方法 经限制性内切酶和序列分析,用脂质体转染技术将其转入B16细胞,G418稳定筛选后IFA法检测其表达,RT-PCK法检测HPV16E6E7mRNA的生成,并将转染细胞接种小鼠皮下,观察成瘤情况.结果 酶切鉴定证实重组质粒中插入的目的基因片段及载体大小、方向和插入住点均正确,在转染的B16细胞中可见绿色荧光并检测到HPV16E6E7mRNA的生成,接种的转染细胞在小鼠皮下100%成瘤.结论 提示B16细胞转染E6E7后其致瘤性与转染空载体组和野生型B16细胞组无明显差异.     

HPV16/18和HPV16E6/E7DNA在宫颈癌组织中的表达

目的检测HPV16/18和HPV16E6/E7 DNA在宫颈癌组织中的表达,探讨其在宫颈癌发病中的作用.方法应用PCR和琼脂糖凝胶电泳方法检测46例宫颈癌组织中HPV16/18和HPV16E6/E7DNA.结果 46例宫颈癌中56.5%(26/46)扩增HPV16/18 DNA,其中宫颈鳞癌25例,宫颈腺癌1例.正常对照组20例HPV16/18DNA均为阴性,与宫颈癌组相比差异有显著性(P<0.01).HPV16/18 DNA阳性拷贝对数值为4.32±2.45.HPV16E6,E7DNA分别有53.8%(14/26)、46.2%(12/26)扩增.结论 HPV16/18和HPV16E6/E7 DNA与宫颈癌的发生密切相关,是宫颈癌恶性转化的关键之一,预示着宫颈癌有较强的增殖能力和转移能力.     

Wnt signaling in Alzheimer's disease: Up or down, that is the question

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by amyloid-β (Aβ) plaques and hyperphosphorylated tau accumulation. AD occurs sporadically (SAD), or is caused by hereditary missense mutations in the amyloid precursor protein (APP) or presenilin-1 and -2 (PSEN1 and PSEN2) genes, leading to early-onset familial AD (FAD). Accumulating evidence points towards a role for altered Wnt/β-catenin-dependent signaling in the etiology of both forms of AD. Presenilins are involved in modulating β-catenin stability; therefore FAD-linked PSEN-mediated effects can deregulate the Wnt pathway. Genetic variations in the low-density lipoprotein receptor-related protein 6 and apolipoprotein E in AD have been associated with reduced Wnt signaling. In addition, tau phosphorylation is mediated by glycogen synthase kinase-3 (GSK-3), a key antagonist of the Wnt pathway.In this review, we discuss Wnt/β-catenin signaling in both SAD and FAD, and recapitulate which of its aberrant functions may be critical for (F)AD pathogenesis. We discuss the intriguing possibility that Aβ toxicity may downregulate the Wnt/β-catenin pathway, thereby upregulating GSK-3 and consequent tau hyperphosphorylation, linking Aβ and tangle pathology. The currently available evidence implies that disruption of tightly regulated Wnt signaling may constitute a key pathological event in AD. In this context, drug targets aimed at rescuing Wnt signaling may prove to be a constructive therapeutic strategy for AD.     

HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6

Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.     

pcDNA3/HPV16 E6真核表达质粒的构建及裸DNA注射动物实验观察

目的探讨ＨＰＶ１６Ｅ６基因ＤＮＡ诱发体液和细胞免疫反应的能力。方法利用基因工程技术构建了ＨＰＶ１６Ｅ６真核表达质粒ｐｃＤＮＡ３／Ｅ６，脂质体法转染Ｃｏｓ７细胞，肌肉注射免疫ＢＡＬＢ／ｃ小鼠，免疫组化技术检测抗体产生及抗原表达。结果被转染的Ｃｏｓ７细胞表达ＨＰＶ１６Ｅ６蛋白免疫小鼠产生抗ＨＰＶ１６Ｅ６抗体。结论这一结果为ＨＰＶ１６相关宫颈癌治疗性ＤＮＡ疫苗的研制提供了资料。     

Stabilization of HPV16 E6 protein by PDZ proteins, and potential implications for genome maintenance

The E6 protein from high-risk human papillomaviruses appears necessary for persistence of viral episomes in cells but the underlying mechanism is unclear. E6 has many activities, including its ability to bind and degrade PDZ domain-containing proteins, such as hScrib. However little is known about the role of these interactions for E6 function and the viral life cycle. We now show that the levels of expression of wild-type E6 are increased in the presence of hScrib whilst a mutant E6 protein lacking the PDZ-binding motif is found at lower levels as it is turned over more rapidly by the proteasome. This correlates with an inability of genomes containing this mutation to be maintained as episomes. These results show that E6 association with certain PDZ domain-containing proteins can stabilize the levels of E6 expression and provides one explanation as to how the PDZ-binding capacity of E6 might contribute to genome episomal maintenance.     

HPV58 E6的基因克隆及HLA-DQB1*03限制性T细胞表位分析

目的:克隆HPV58E6的基因,并对其HLA-DQB1*03限制性T细胞表位进行分析。方法:从宫颈癌组织中提取基因组DNA,通过PCR方法扩增HPV58E6基因,常规方法将目的基因插入pGEM-TEasy载体中。酶切和测序鉴定后,利用位置特异性分数矩阵(PSSM)理论、支持向量机(SVM)理论和蛋白酶体酶切位点预测算法分析HPV58E6的HLA-DQB1*03限制性T细胞表位。结果:成功克隆了1株HPV58E6基因(GenBank EF060239),表位47FADLRIAY-RDGNPFA和表位102RCIICQRPLCPQEKK理论上是其HLA-DQB1*03限制性T细胞表位。结论:这两个表位可望作为后续相关实验中表位筛选、鉴定和多肽疫苗研制的候选对象。     

Wnt信号分子在大鼠中枢神经系统的表达及其共存

目的:通过研究Wnt信号分子在大鼠中枢神经系统(CNS)的表达及分布,以探讨Wnt信号分子在CNS早期发育的可能调控机制,以及Wnt信号分子之间的功能联系.方法:应用免疫组织化学及双标记技术,观察了Wnt信号分子β-catenin、糖原合成酶激酶3β(Gsk-3β)、大肠腺瘤样息肉基因(APC)等关键调控分子在成年大鼠CNS的表达分布、细胞定位及共存关系.结果:免疫组织化学显色显示Gsk-3β阳性细胞多为具有突起的神经元样细胞,其主要分布区域包括新皮层、背内侧丘脑、海马、小脑浦肯野细胞及脑干多个核团.β-catenin与APC在分布模式上与Gsk-3β高度一致.双标记实验显示β-catenin与APC除神经元外,在部分星形胶质细胞也存在表达,而Gsk-3β则主要显示神经元定位.部分β-catenin阳性细胞,特别是室下层及海马区阳性细胞还呈现与神经前体细胞标记物巢蛋白的共存.结论:Wnt信号分子在CNS存在着密切相关的功能联系,同时在不同细胞及组织区域可能还具有其特有的生物效应,与神经前体细胞和神经胶质细胞增殖分化密切相关,对其相关信号机制有待深入研究.     

A novel real-time genotyping assay for detection of the E6-350G HPV 16 variant

It has been suggested that some E6 human papillomavirus (HPV) type 16 variants could be involved in viral persistence and progression of HPV infection. A novel one-step allelic discrimination real-time PCR was evaluated for E6-350G variant detection in 102 endocervical HPV 16 positive samples. This assay was also used to assess the distribution of this variant in Spanish women with cervical cancer related to HPV 16.The detection limit for the allelic discrimination assay was 50 copies per reaction, even where the E6-350G variant represents only 20% of the variants in the sample. Complete concordance was observed between DNA sequencing and the novel AD RT-PCR assay. Fourteen E6-350T reference strains and 18 E6-350G variants were detected out of 32 endocervical samples from women with cervical cancer. The average age of women who were infected by the E6-350G HPV 16 variant was 10 years lower in these samples than in women who were infected by the reference strain.This novel allelic discrimination assay is a fast, sensitive and specific method for detection of the E6-350G HPV 16 variant.     

Effectiveness of HPV 16 viral load and the E2/E6 ratio for the prediction of cervical cancer risk among Chinese women

The effectiveness of the E2/E6 ratio, the state of viral genome integration and the viral load of human papillomavirus 16 (HPV 16) in predicting the risk of cervical cancer among Chinese women was investigated. Quantitative PCRs for the E2/E6 ratio and the viral load were performed on 85 cervical cancer samples and 55 HPV 16 positive healthy controls. The integrated form of the viral genome was found in 10.9% control samples and in 26.4% cervical cancer samples (P = 0.02). The majority of the cervical cancer (63.2%) and control samples (60%) were mixed forms. The E2/E6 ratio was associated with a high risk of cervical cancer (OR = 7.29, P = 9.55E?6). The integrated form (OR = 6.54, P = 0.005) and mixed form (OR = 2.93, P = 0.042) increased the risk of cervical cancer. The mean viral load in cervical cancer samples (37,371 ± 227,135) was higher than that in the controls (4,619 ± 27,079; P = 0.011). Additionally, the viral load increased along with the cervical cancer progression from the International Federation of Gynecology and Obstetrics (FIGO) stage I (12,337 ± 25,604) to stage II (67,453 ± 319,821). Compared with the state of viral genome integration (area under the receiver operating characteristic curve (AUC) = 0.743) or the viral load (AUC = 0.694), the E2/E6 ratio improved the effectiveness of the risk prediction of cervical cancer (AUC = 0.777), with the sensitivity (specificity) 81.2% (71.7%). The state of viral genome integration and the viral load of HPV 16 were important factors for the risk prediction of cervical cancer among Chinese women, and the E2/E6 ratio had a better cervical cancer risk prediction with age adjustment. J. Med. Virol. 85:646–654, 2013. © 2013 Wiley Periodicals, Inc.     

人乳头瘤病毒16型修饰后E6E7基因免疫原性增强

利用突变修饰后消除转化活性并保留抗原性的中国山东地方株人乳头瘤病毒16型(human papillomavirus type 16,HPVl6)E6E7融合基因(fmE6E7)，研制治疗HPVl6相关疾病的DNA疫苗。用PCR扩增fmE6E7基因后，插人真核表达质粒获得pVRl012-fmE6E7，瞬时转染Cos-7细胞，免疫荧光法检测证实其表达后，在C57BL／6小鼠后腿肌肉进行裸DNA免疫，5lCr释放法体外分析免疫鼠的细胞毒性T淋巴细胞活性Cytotoxic T lymphocyte，CTL)，间接ELISA法检测免疫鼠血清中E7特异性抗体。研究表明修饰后的中国地方株E6E7融合基因可诱导机体产生特异的抗体反应和CTL反应，与单独野生型E7基因免疫相比，E6E7融和基因可更好的活化CTT反应。表明修饰后消除转化活性的中国地方株E6E7融合基因可作为HPVl6治疗性DNA疫苗的靶基因。     

重组人乳头瘤病毒16型E6蛋白的表达、纯化和动物免疫效果分析

目的 诱导表达人乳头瘤病毒16型(HPV16)E6蛋白,制备可溶性蛋白,并通过动物免疫进行免疫效果评估.方法 采用IPTG诱导pQE30-HPV16E6/BL21(DE3)重组菌株,表达产物经SDS-PAGE和Western blot分析鉴定.提取包涵体进行变性处理,变性蛋白经Ni2+金属螯合层析纯化,将纯化产物用复性透析方法处理以获取可溶性蛋白.免疫Bal B/c小鼠,检测血清抗体、CD4+/CD8+和IFN-γ.结果 诱导后重组菌有相对分子量18 000蛋白质表达,以不溶性包涵体为主要表达方式.目的蛋白可与HPV16E6多克隆抗体识别,纯化和透析处理后得到可溶性的蛋白.小鼠免疫后血清抗体滴度升高,淋巴细胞CD4+/CD8+升高,IFN-γ未见升高.结论 成功表达了HPV16E6蛋白,同时处理包涵体并制备了可溶性目的蛋白.目的蛋白可刺激小鼠体内产生有效的免疫反应,该蛋白的成功制备为患者血清抗体的检测和肿瘤细胞杀伤的免疫研究奠定了坚实基础.     

子宫颈癌、子宫颈上皮内病变组织中SIRT1、HPV 16/18E6的表达及其意义

目的检测子宫颈癌及癌前病变中SIRT1的表达情况,探讨其与临床病理特征的关系。同时检测早期癌蛋白中HPV16/18E6的表达,分析两者的相关性。方法应用免疫组化En Vision法检测30例子宫颈炎、100例子宫颈上皮内病变(高级别、低级别各50例)、30例子宫颈鳞状细胞癌组织中SIRT1和HPV 16/18E6的表达。结果子宫颈癌组织中SIRT1阳性率为93.33%(28/30),高于子宫颈炎组织(13.33%,4/30),差异有统计学意义(P0.05)。子宫颈高级别上皮内病变SIRT1阳性率(88%,44/50)高于低级别上皮内病变(14%,7/50),差异有统计学意义(P0.05)。但高级别上皮内病变与子宫颈癌SIRT1阳性率差异无统计学意义(P0.05);低级别上皮内病变与子宫颈炎中SIRT1阳性率差异无统计学意义(P0.05)。在子宫颈癌中,SIRT1的阳性率与临床分期和组织学分级有关,晚期、分化差的子宫颈癌阳性率高于早期、分化好的子宫颈癌,差异有统计学意义(P0.05)。在子宫颈癌中SIRT1的阳性率(93.33%,28/30)高于HPV 16/18E6(30%,9/30),在子宫颈高级别上皮内病变中SIRT1的阳性率(88%,44/50)高于HPV 16/18E6(28%,14/50),但在低级别上皮内病变中SIRT1的阳性率(14%,7/50)低于HPV 16/18E6(36%,18/50),两者差异有统计学意义(P0.05)。结论 SIRT1的阳性率随着病变程度增加而升高,提示SIRT1与细胞恶性转变有关,其过表达可能促进上皮向高级别内病变乃至子宫颈癌进展。