人乳头瘤病毒16型E6E7基因反义质粒对人宫颈癌细胞恶性的逆转…

构建可为地塞米松诱导表达的人乳头瘤病毒１６型Ｅ６Ｅ７，基因反义质粒，利用磷酸钙沉淀法将其分别转染到ＨＰＶ－１６阳性的人宫颈癌株Ｃａｓｋｉ和ＨＰＶ阴性的人宫颈癌细胞株Ｃ－３３Ａ中。地塞米松诱导反义质粒表达后，ＣａｓＫｉ细胞失去其恶性表型，而Ｃ－３３１细胞的生长特性及恶性行为未发生变化。     

人乳头瘤病毒16型E6E7反义RNA抑制宫颈癌细胞恶性表型的作用

目的研究癌基因的特异性反义ＲＮＡ对癌细胞生长繁殖和恶性程度的影响。方法用逆转录病毒载体将人乳头瘤病毒（ＨＰＶ）－１６Ｅ６Ｅ７反义ＲＮＡ导入ＨＰＶ－１６ＤＮＡ阳性的宫颈癌细胞株ＣａＳｋｉ中，观察该细胞在导入反义ＲＮＡ后其表型特征和在裸鼠体内致癌能力的变化。结果ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能降低宫颈癌细胞ＣａＳｋｉ的生长速率，抑制其在软琼脂上的集落形成能力，并能明显地抑制其在裸鼠体内的致癌能力。Ｗｅｓｔｅｒｎｂｌｏｔ分析发现ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能使宫颈癌细胞中病毒ＨＰＶ－１６Ｅ６基因的表达水平降低。结论ＨＰＶ－１６Ｅ６Ｅ７反义ＲＮＡ能使宫颈癌细胞ＣａＳｋｉ恶性表型逆转；由其引起的癌细胞中ＨＰＶ－１６癌基因表达水平的降低可能是癌细胞表型逆转的原因之所在；ＨＰＶ－１６癌基因的表达水平对维持癌细胞的恶性表型起着重要作用。     

人乳头瘤病毒16型E6E7反义RNA抑制宫颈癌细胞恶性？…

目的 研究癌基因的特异性反义ＲＮＡ对癌细胞生长繁殖和恶性程度的影响。方法 用逆转录病毒载体将人乳头瘤病毒（ＨＰＶ）－６Ｅ６Ｅ７反义ＲＮＡ导入ＨＰＶ－１６ＤＮＡ阳性的宫颈癌细胞株ＣａＳｋｉ中，观察该细胞在导入反义ＲＮＡ后其表型特征和在裸鼠体内致癌能力的变化。结果 ＨＰＶ－１６ Ｅ６Ｅ７反义ＲＮＡ能降低宫颈癌细胞ＣａＳｋｉ的生长速率，抑制其在软琼脂上的集落形成能力，并能明显地抑制其在裸鼠体内的致癌能力     

人乳头瘤病毒16 E6蛋白单克隆抗体的研制

运用杂交瘤技术，我们成功地建立了两株能稳定分泌小鼠抗人乳头瘤病毒１６Ｅ６蛋白单克隆抗体的杂交瘤细胞。经鉴定单克隆抗体均属ＩｇＧ１ｋ。试验结果表明，所得单克隆抗体仅与ＨＰＶ１６Ｅ６融合蛋白反应，不与ＨＰＶ１６Ｅ７、Ｌ１、Ｌ２融合蛋白以及Ｌ１－Ｌ２真核表达蛋白反应，也只与Ｃａｓｋｉ细胞反应，不与Ｈｅｌａ细胞反应。初步结果说明，该抗体是ＨＰＶ１６Ｅ１６蛋白特异性的ＭｃＡｂ。     

人乳头瘤病毒16E6蛋白单克隆抗体的研制

运用杂交瘤技术，我们成功地建立了两株能稳定分泌小鼠抗人乳头瘤病毒１６Ｅ６蛋白单克隆抗体的杂交瘤细胞，经鉴定单克隆抗体属ＩｇＧ１ｋ。试验结果表明，所得单克隆抗体仅１与ＰＶ１６Ｅ６融合蛋白反应。不与ＨＰＶ１６Ｅ７、Ｌ１、Ｌ２融合蛋白以及Ｌ１－Ｌ２真核表达蛋白反尖，也只与Ｃａｓｋｉ细胞反应，不与Ｈｅｌａ细胞反应，初步结果说明，该抗体是ＨＰＶ１６Ｅ１６蛋白特异性的ＭｃＡｂ。     

人乳头瘤病毒16型E6E7 ORF转化作用的研究

构建了含人乳头瘤病毒１６型（ＨＰＶ１６）－Ｅ６Ｅ７ＯＲＦｓ（ｎｔ８３－８５５）片段和ＨＰＶ１６长控制区（ＬＣＲ）加Ｅ６Ｅ７ＯＲＦｓ片段（ｎｔ７００７－７９０４／０－８７９）的逆转录病毒载体ｐＨ２１和ｐＨ１８质粒，利用Ｌｉｐｏｆｅｃｔｉｎ分别将它们导入病毒包装细胞ｐＡ３１７中，经过筛选获得Ｇ４１８抗性的病毒包装细胞，产生的重组病毒Ｈ２１和Ｈ１８感染的ＮＩＨ３Ｔ３细胞都具有恶性细胞的形态学特征，并能在裸鼠体内形成肿瘤。Ｓｏｕｔｈｅｒｎ杂交结果证明，上述两基因片段都整合到细胞基因组中。本实验结果说明ＨＰＶ１６－Ｅ６Ｅ７基因片段是ＨＰＶ１６转化ＮＩＨ３Ｔ３细胞的关键早期区，其自身ＬＣＲ区在该转化过程中没有显示出重要作用。     

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。     

中国地方株人乳头瘤病毒16型E7基因一级结构及其变异

从湖北地区一宫颈癌活检组织中提取ＤＮＡ，采取加端聚合链反应（Ａｄｄ－ｏｎ ＰＣＲ）技术，获得了人乳头瘤病毒１６型E７基因（ＨＰＶＩ６Ｅ７）。将该基因克隆于载体ｐＵＣ１８后，进行了该基因一级结构顺序分析。完整的ＨＰＶ１６Ｅ７湖北株基因（ＨＰＶ１６Ｅ７－ＨＢ）全长２９４ｂｐ，与已发表的德国株（ＧＳ）大小一致，但其核苷酸顺序中有２处发生了变异，均为Ｃ→Ｔ变异。第４３位ＣＡＡ→ＴＡＡ使相应的谷氨酰胺密码子变为终止密码，形成无义突变（Ｎｏｎｓｅｎｓｅｍｕｔａｔｉｏｎ）。将重组质粒中０．３ｋｂ的ＨＰＶ１６Ｅ７基因在表达载体ＰＷＲ５９０－１中进行克隆，经诱导使重组表达质粒在大肠杆菌中高效表达，得到了预计的、分子量约为６９×１０~３的融合蛋白。该蛋白的表达量占菌体总蛋白量的３０％左右。该试验表明ＨＰＶ１６Ｅ７一级结构以及所编码的蛋白多肽在不同的国家和／或不同的地区可能存在着差异。本文首次报道了中国地方株ＨＰＶ１６Ｅ７基因的一级结构。     

宫颈癌组织中人乳头瘤病毒16型E7蛋白致癌机理初探

目的 研究宫颈癌组织中人乳头瘤病毒（ＨＰＶ）１６－Ｅ７蛋白对视网膜母细胞瘤基因（Ｒｅｔｉｎｏｂｌａｓｔｏｍａ）Ｒｂ蛋白及Ｅ２Ｆ－１的作用的机制,探讨ＨＰＶ１６－Ｅ７蛋白与宫颈癌发生的关系。方法 采用聚合酶链反应检测宫颈癌及正常宫颈组织中ＨＰＶ１６感染等,用蛋白印迹技术对ＨＰＶ１６ ＤＮＡ阳性的宫颈癌组织中是否存在ＨＰＶ１６－Ｅ７蛋白和Ｒ６蛋白－Ｅ２Ｆ－１形成的复合物进行检测。正常宫颈组织作为对照,     

中国地方株人乳头瘤病毒16型E7基因一级结构及其…

从湖北地区一宫颈癌活检组织中提取ＤＮＡ，采取加端聚合酶链反应（ＰＣＲ）技术，获得了人乳头瘤病毒１６型Ｅ７基因（ＨＰＶ１６Ｅ７）。将该基因克隆于载体ｐＵＣ１８后，进行了该基因一级结构顺序分析。完整的ＨＰＶ１６Ｅ７湖北株基因（ＨＰＶ１６Ｅ７－ＨＢ）全长２９４ｂｐ，与已发表的德国株（ＧＳ）大小一致，但其核苷酸顺序中有２处发生了变异，均为Ｃ→Ｔ变异。第４３位ＣＡＡ→ＴＡＡ使相应的谷氨酰胺密码子变为终止密码     

特异siRNA抑制宫颈癌细胞中人乳头状瘤病毒16型E6表达的研究

目的 优化HPV-16 E6癌基因特异的U6质粒表达的siRNA，抑制HPV癌基因表达及其对子宫颈癌细胞生长繁殖的影响。方法 选择4个分别针对HPV-16 E6 mRNA外显子和内含子序列为靶序列，合成DNA链，构建表达HPV-16 E6短发卡样dsRNA的重组pSilencer1．0-U6载体，导入HPV-16DNA阳性的宫颈癌细胞株CaSki中，观察该细胞中HPV-16 E6、E7基因表达水平及其蛋白含量的变化，并观察细胞生长被抑制的情况。结果 4种HPV-16 E6 siRNA均能降低宫颈癌细胞CaSki的生长速率。通过细胞生长曲线观察到HPV-16 E6 shRNA表达质粒导入细胞0-96h内，可降低细胞生长速度。荧光定量RT-PCR检测HPV-16 E6 siRNA可使宫颈癌细胞株CaSki中HPV-16 E6、E7基因转录的mRNA水平降低，其中针对E6 mRNA内含子的重组shRNA只抑制E6基因的表达水平。Western blot分析表明，4个HPV-16 E6 siRNA作用72h后，未能检测到宫颈癌细胞中HPV-16 E6蛋白。结论 HPV-16 E6 siRNA能使宫颈癌细胞CaSki生长缓慢；选择针对E6内含子的siRNA作用位点，特异性抑制E6表达；而针对E6外显子的siRNA作用位点，可抑制E6和E7基因的表达，是用于治疗HPV阳性宫颈癌细胞的理想靶位。     

西多福韦对人宫颈癌细胞CaSki内人乳头瘤病毒16抑制作用研究

目的从抗病毒角度探讨西多福韦对宫颈癌细胞 CaSki内人乳头瘤病毒16（HPV16）的抑制作用及对细胞周期的影响。方法用 MTT法检测西多福韦对细胞的毒性;用实时定量 PCR法检测其对病毒 E6、E7 mRNA水平的影响;用 Western blot方法检测其对病毒蛋白 E6、E7和细胞抑癌蛋白 p53、pRb表达水平的影响;用流式细胞法检测其对宫颈癌细胞周期的影响。结果西多福韦对宫颈癌细胞毒性较正常细胞大。可使HPV16阳性宫颈癌细胞 CaSki内 E6、E7 mRNA和蛋白水平降低,最大抑制率分别为（33.38±8.00）%、（28.32±2.73）%和98.92%、97.46%;可以使 p53、pRb蛋白水平升高,最大浓度时可以上调12.06和3.53倍;对 HPV16阴性宫颈癌细胞 C-33A p53蛋白表达无影响,但可提高 pRb蛋白水平;可导致 CaSki和 C-33A细胞发生 S期阻滞,最高浓度组细胞相对对照组 S期分别增加22.83%和67.64%。结论西多福韦可以在对细胞无毒的浓度下,抑制宫颈癌细胞内的 HPV16,诱导宫颈癌细胞发生 S期阻滞。     

Cervical keratinocytes containing stably replicating extrachromosomal HPV-16 are refractory to transformation by oncogenic H-Ras

Ras expression in human epithelial cells with integrated HPV genomes has been shown to cause tumorigenic transformation. The effects of Ras in cells representing early stage HPV-associated disease (i.e., when HPV is extrachromosomal and the oncogenes are under control of native promoters) have not been examined. Here, we used human cervical keratinocyte cell lines containing stably replicating extrachromosomal HPV-16 and present the novel finding that these cells resist transformation by oncogenic H-Ras. Ras expression consistently diminished anchorage-independent growth (AI), reduced E6 and E7 expression, and caused p53 induction in these cells. Conversely, AI was enhanced or maintained in Ras-transduced cervical cells that were immortalized with a 16E6/E7 retrovirus, and minimal effects on E6 and E7 expression were observed. Ras expression with either episomal HPV-16 or LXSN-E6/E7 was insufficient for tumorigenic growth suggesting that other events are needed for tumorigenic transformation. In conclusion, our results indicate that Ras-mediated transformation depends on the context of HPV oncogene expression and that this is an important point to address when developing HPV tumor models.     

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.     

Antibodies to HPV-16 E6 and E7 proteins as markers for HPV-16-associated invasive cervical cancer.

Transforming proteins E6 and E7 of human papillomaviruses (HPVs) are consistently expressed in HPV-associated cervical cancers. In ELISA with four HPV-16 E6-E7 peptides, patients with HPV-16-associated invasive cervical cancer (group 1) had a greater seroreactivity than all other groups, which included patients with HPV-16-associated cervical intraepithelial neoplasia, invasive cervical cancer patients without HPVs, and unaffected controls. A larger proportion of group 1 sera, as compared to sera of all other groups, was reactive with at least one peptide (49% vs 17-27%), and with two or more peptides (22% vs 0-6%). A clear difference between group 1 and all other groups was also found for high ELISA absorbance values to at least one peptide (22% vs 0-8%). This high seroreactivity of group 1 sera was confirmed by a radioimmunoprecipitation assay with in vitro transcribed and translated HPV-16 E7 protein. Sera from 50% of group 1 but only 3% of controls were reactive in this test. Antibodies to HPV-16 E6 and E7 proteins appear to be virus-specific and disease state-specific markers of HPV-associated cervical cancer.     

Codon optimization of the HPV-16 E5 gene enhances protein expression

The human papillomavirus type 16 (HPV-16) E5 protein is an 83-amino-acid, hydrophobic polypeptide that has been localized to intracellular membranes when overexpressed in COS-1 cells. While the HPV-16 E5 protein appears to modulate endosomal pH and signal transduction pathways, genetic analysis of its biological activities has been hampered by low (usually nondetectable) levels of expression in stable cell lines. Sequence analysis of the native HPV-16 E5 gene revealed that infrequent-use codons are used for 33 of its 83 amino acids and, in an effort to optimize E5 expression, we converted these codons to those more common in mammalian genes. The modified gene, 16E5*, generated protein levels that were six- to ninefold higher than those of wild-type HPV-16 E5, whereas the levels of mRNA were unchanged. 16E5* protein was detectable in keratinocytes by immunoblotting, immunoprecipitation, and immunofluorescence techniques and formed disulfide-dependent dimers and higher-order oligomers. Unlike the bovine papillomavirus E5 protein, which is present in the Golgi, 16E5* was localized primarily to the endoplasmic reticulum and its expression reduced the in vitro life span of keratinocytes.