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型E＿6E＿7基因反义质粒对人宫颈癌细胞恶性的逆转作用

构建可为地塞米松诱导表达的人乳头瘤病毒１６型（ＨＰＶ－１６）Ｅ＿６Ｅ＿７基因反义质粒（ｐ１６ａｓＥ＿６Ｅ＿７Ｎｅｏ），利用磷酸钙沉淀法将其分别转染到ＨＰＶ－１６阳性的人宫颈癌细胞株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－３３Ａ细胞的生长特性及恶性行为未发生变化。说明反义质粒能够改变Ｃａｓｋｉ细胞的恶性表型，且这种改变是通过特异性抑制Ｅ＿６Ｅ＿７基因表达实现的。     

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 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。     

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。     

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。     

人乳头状瘤病毒18型E6E7基因诱导人胚食管上皮永生化

目的为研究病毒和肿瘤的关系,用人乳头状瘤病毒（ＨＰＶ）１８型Ｅ６Ｅ７基因感染胎儿食管上皮,建立一株新的人食管上皮永生化细胞株（ＳＨＥＥ）。方法ＨＰＶ１８Ｅ６Ｅ７腺病毒伴随病毒（ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ））载体的构建;胚胎食管组织培养,ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ感染,继续培养传代。用光镜、电镜检查其形态;聚合酶链反应（ＰＣＲ）、荧光原位杂交（ＦＩＳＨ）检查该病毒片段;用软琼脂培养和裸鼠接种检查致瘤性。结果经过长时间的传代培养,ＳＨＥＥ的表型仍保留原代上皮细胞培养的特征,表现为单层生长和锚锭依赖性生长,在软琼脂培养不形成克隆,接种裸鼠未成瘤。ＳＨＥＥ细胞系电镜检查可见张力原纤维,免疫组织化学检查细胞角质蛋白阳性,证实为鳞状上皮来源。ＦＩＳＨ和ＰＣＲ检测显示有ＨＰＶ１８Ｅ６Ｅ７基因。结论用ＨＰＶ１８Ｅ６Ｅ７基因建立食管上皮永生化细胞株ＳＨＥＥ,支持ＨＰＶ１８可能和食管癌病因有关的观点,可进一步用以研究食管癌的病因和发病机制。     

斑点杂交及RNA酶保护分析法检测反义HSP90基因转染细胞 …

目的：检测ＨＳＰ９０反义核酸转染细胞后反义ＲＮＡ的表达,方法：采用打点杂交及ＲＮＡ酶保护分析法,结果：ＨＳＰ９０反义核酸转染细胞ＡＨ－ＳＧＣ７９０１,ＡＨ－ＳＧＣ７９０１／ＶＣＲ,ＡＨ－ＨＣＣ７４０２及ＡＨ－Ｅｃ１０９有ＨＳＰ９０反义ＲＮＡ的表达。结论：ＥＳＰ９０反义ＲＮＡ在ＡＨ－ＳＧＣ７９０１,ＡＨ－ＳＧＣ７９０１／ＶＣＲ,ＡＨ－ＨＣＣ７４０２及ＡＨ－Ｅｃ１０９细胞的表达,为进一步研究ＨＳＯ９０     

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,     

Genus specific features of bovine papillomavirus E6, E7, E5 and E8 proteins

Six bovine papillomavirus (BPV) types, BPV-1 to -6, have been classified in genera Delta-papillomavirus (BPV-1 and -2), Epsilon-papillomavirus (BPV-5) and Xi-papillomavirus (BPV-3, -4 and -6). In addition, 16 unclassified putative BPV types have been reported. In the present study, we characterized genus specific features of E6, E7, E5 (formerly E8) and E8 proteins of seven putative BPV types, BAPV-1, -2, -3, -4 and -10, BAA-5 and BPV-3c. These putative BPV types were classified in genera Epsilon- or Xi-papillomavirus. The E6 proteins of BPV and putative BPV types in Epsilon-papillomavirus showed high sequence similarities, and contained two typical zinc-binding domains. However, E7 proteins contained atypical zinc-binding domains, and lacked the canonical retinoblastoma tumor suppressor protein (pRB)-binding motif. BPV and putative BPV types in Xi-papillomavirus contained E5 or E8 open reading frame (ORF) in the E6 position. The E5 ORFs encoded proteins consist of 42-amino acid with a hydrophobic transmembrane and a hydrophilic C-terminal domain. But the E8 ORFs encoded protein which have two transmembrane domains. Our results demonstrated that E5, E8, E6, E7 proteins of the putative BPV types, which are presumably classified in genera Epsilon- or Xi-papillomavirus, retained the some genus specific features.     

Phage Typing Set for E1, E2, E3, and E4 Salmonellae

Eighteen common serotypes representative of group E₁, E₂, E₃, and E₄Salmonella were characterized using a single set of phages.     

(E)TUDE APPLIQ(E)E DE ENC(E)PHALE ATLAS DANS MALADES AVEC INFARCTION C(E)R(E)BRALE

目的:探讨脑电地形图在脑梗死中的应用价值。方法:在43例脑梗死患者中,进行了脑电地形图和脑CT检查,并进行了对比。结果:脑电地形图和脑CT的改变是一致的,但脑电地形图改变早于脑CT。结论:脑电地形图在脑梗死患者中有重要的应用价值。     

Non-apolipoprotein E and apolipoprotein E genetics of sporadic Alzheimer's disease

The genetic epidemiology of sporadic Alzheimer's disease (SAD) remains a very active area of research, making it one of the most prolifically published areas in medicine and biology. Numerous putative candidate genes have been proposed. However, with the exception of apolipoprotein E (APOE), the only confirmed genetic risk factor for SAD, all the other data appear to be not consistent. Nevertheless, the genetic risk for SAD attributable to the APOE gene in the general population is 20–70%, providing a strong evidence for the existence of additional genetic risk factors. The first part of the present article was dedicated to non-APOE genetics of SAD, reviewing chromosomes-by-chromosomes the available data concerning the major candidate genes. The second part of this article focused on some recently discovered aspects of the APOE polymorphism and their implications for SAD. An attempt to identify the future directions for non-APOE genetic research in SAD was also discussed.     

Molecular variants of human papilloma virus 16 E2, E4, E5, E6 and E7 genes associated with cervical neoplasia in Romanian patients

The aim of this study was to identify and associate the sequence variations of human Papillomavirus 16 (HPV16) genes from women who live in two different areas of Romania and associate them with malignant progression. One hundred twenty-four HPV16-positive cervical isolates were collected, and the E2, E4, E5, E6 and E7 viral genes were sequenced. Two new missense mutations in the E6 gene (C279G and A305C) were found (together or alone, in association with other mutations) in 44 of 124 cases. The most frequently simultaneously mutated genes were E4/E2 hinge, E5 and E6 (p = 0.0004) in squamous cell carcinoma (SCC) samples. Also, for SCC patients, the best-correlated mutation patterns were obtained for E4/E2 hinge-E5 (r = 0.7984; p < 0.0001). No sample was found to have all of the investigated viral genes concurrently mutated. Phylogenetic analysis was performed to characterize the viral variants. Similar results were found for SCC and cervical intraepithelial neoplasia III (CINIII) cases. After all of the target gene sequences were assembled, all patients were found to be infected with viruses of the HPV16- European-German (EG) lineage, and two clusters were identified, the first (55/96 variants) from Moldavia and the second (41/96 variants) from Bucharest. The distinct cluster derived from EG in Moldavia could partially explain the increased frequency of SCC in this area. This study has generated a comprehensive set of sequence variation data on HPV16 circulating in Romania to join the existing data and highlight the important role of HPV16 variants during cervical carcinogenesis.     

Apolipoprotein E deficiency associated with type III hyperlipoproteinemia--analysis of apolipoprotein E

Case report: The patient was a 50-year old male who was found to have a high cholesterol level during a routine health check up at work 5 years before and was examined at Keio University Hospital. Lipoprotein electrophoresis on agarose gel revealed type III hyperlipidemia, and a screening test yielded the following values (mg/dl): total cholesterol, 420; TG, 138; and HDL-cholesterol, 105. Turbidimetric immunoassay showed that the apolipoprotein E (apoE) level was below the limit of detection. Since he was 25 years old, the patient had sometimes noticed xanthomas on his knees and eyelids, and for that reason we made a diagnosis of apoE deficiency associated with type III hyperlipidemia. We tried using SDS-polyacrylamide gel electrophoresis, Western blot, and the protein chip method to detect apoE in this case, but the level was below the limit of detection by the first two methods, and it was so low that it was detected near the sensitivity limit of the protein chip method. Diet therapy, statin therapy, and fibrate therapy have been continued, and the latest data are: total cholesterol, 373; TG, 95; and HDL-cholesterol, 83. No manifestations associated with arteriosclerotic disease other than mild xanthomas have been observed.