人乳头状瘤病毒18型E6E7基因诱导胎儿食管永生化上 …

目的 ＳＨＥＥ细胞系是经人乳头状瘤病毒（ＨＰＶ）１８型Ｅ６Ｅ７基因诱导的永生化上皮细胞株,已传代超过５０代。研究胎儿食管上皮永生化的细胞 ＳＨＥＥ生物学特性,包括增殖,分化和调亡。方法 细胞于１９９培养基培养,用光镜、电镜和荧光显微镜研究其生长率、形态和染色体分析;用流式细胞仪研究其细胞增殖动力学;用免疫组织化学方法研究Ｋｉ６７和角蛋白和用末端转移酶标记（ＴＵＮＥＬ）凋亡细胞。结果 细胞培养呈单导     

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。     

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     

人乳头状瘤病毒16型E6E7肿瘤性转化人永生化支气管上皮细胞

目的 探讨人乳头状瘤病毒（ＨＰＶ）１６型Ｅ６Ｅ７片段对人永生化支气管上皮细胞系ＴＲ细胞的作用。方法 将Ｅ６Ｅ７片段构建入逆转录病毒载体,导入ＴＲ细胞,观察生长特性和致瘤性的改变;并用免疫沉淀（ＩＰ）－Ｗｅｓｔｅｒｎ ｂｌｏｔ检测ｐ２７蛋白功能及ＦＡＫ、桩蛋白数量及磷酸化状况,结果 嘌呤霉素抗药性克隆ＴＲ／Ｅ６Ｅ７有Ｅ６Ｅ７的存在和稳定表达;ＴＲ／Ｅ６Ｅ７细胞系细胞生长加快,软琼脂集落形成能力增强,     

人乳头状瘤病毒感染与食管癌的关系

人类乳头状瘤病毒（ＨＰＶ）的感染是上皮性肿瘤发生的一个重要原因。特别是ＨＰＶ１６和ＨＰＶ１８型感染与宫颈癌的发生。但ＨＰＶ的感染与食管癌的关系仍不清楚。我们采用免疫组织化学和敏感的ＨＰＶ共同引物、ＨＰＶ１６和ＨＰＶ１８型特异性引物的ＰＣＲ方法及３２Ｐ标记特异性探针Ｓｏｕｒｈｅｒｎ杂交检测食管癌中的ＨＰＶ及其亚型。结果显示，１２７例食管鳞癌标本免疫组化方法检测，ＢＰＶ阳性者占６０．６（７７／１２７），ＨＰＶＥ６蛋白抗原阳性者占４３％（５４／１２７）。其阳性染色明确定位于鳞癌组织内，并与肿瘤的分化有着密切的关系。经β球蛋白引物扩增证实不含ＰＣＲ抑制标本共１０３例，其中ＨＰＶ共同引物ＰＣＲ扩增阳性者为３７例，占３５．９％，经型特异ＰＣＲ及Ｓｏｕｔｈｅｒｎ杂交证实其中ＨＰＶ１６阳性者２１例，占２０．４％；ＨＰＶ１８阳性者８例，占７．８％。仅一例为ＨＰＶ１６和１８均阳性。以上结果显示ＨＰＶ感染的确存在于食管癌中，并可能在食管癌发生中起一定的作用。     

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。     

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。     

两种抗人乳头瘤病毒核酶的设计表达与鉴定

人乳头瘤病毒（Ｈｕｍａｎｐａｐｉｌｌｏｍａｖｉｒｕｓ,ＨＰＶ）是人类肿瘤病毒病因中重要的一类ＤＮＡ病毒,高危ＨＰＶ易引起宫颈癌等恶性肿瘤,其中最常见的是ＨＰＶ１６型和ＨＰＶ１８型,而Ｅ６、Ｅ７基因是主要的致癌基因。根据核酶（Ｒｉｂｏｚｙｍｅ）能序列特异地识别和切割靶ＲＮＡ的特性,分别设计了切割ＨＰＶ１６Ｅ６ｍＲＮＡ和ＨＰＶ１８Ｅ６ｍＲＮＡ的两种核酶（简称抗１６ＨＲｚ、抗１８ＨＲｚ）,并进行了原核克隆、表达与体外活性鉴定;从而证实所设计的核酶具有较高的活性,可作为对ＨＰＶ相关性肿瘤进行基因治疗的…     

A comparative study of telomerase activity and malignant phenotype in multistage carcinogenesis of esophageal epithelial cells induced by human papillomavirus.

To examine certain characteristics of multistep carcinogenesis, we studied telomerase activity and malignant phenotypes in the immortal, premalignant and malignant stages of esophageal epithelial cells induced by HPV. An immortalized human fetal esophageal epithelial cell line (SHEE) was induced by E6E7 genes of human papillomavirus (HPV) type 18. Cells in the 10th passage, (SHEE10), 31st passage (SHEE31), 61st passage (SHEE61) and SHEE61A which were selected and expanded from anchorage-independent growth colonies of SHEE61, were examined as follows: cell morphology by electron-microscopy; the cell cycle by flow cytometry, telomerase activity by TRAP assay, tumorigenic detection including anchorage-independent growth by soft agar culture and tumor formation by inoculating cells into SCID and nude mice, and detection of HPV18 E6E7 oncoprotein by Western blot. The morphology of the SHEE10 cells exhibited good differentiation, the SHEE60 and SHEE61A cells were relatively poorly differentiated, and the SHEE31 cells were differentiated in two distinct ways. The telomerase was activated in SHEE31, SHEE61 and SHEE61A, but not in SHEE10 cells. SHEE61 and SHEE61A cells were weakened in contact-inhibition and increased in anchorage-independent growth. Inoculated into SCID and nude mice, the cells of the earlier two passages could not develop tumors; the SHEE61 developed one tumor in four SCID mice, but not in nude mice, and the SHEE61A cells developed tumors in both strains of immunodeficient mice. HPV18 E6E7 DNA detection by Western blotting was positive in all cell passages. In the process of carcinogenesis by HPV, the cells of SHEE31 are in an immortalized state with telomerase activity. The fact that SHEE61 cells remained immortalized and also demonstrated anchorage-independent growth, reveals premalignant character; the cells of SHEE61A exhibited malignant transformation with tumor formation in mice. The results revealed that the telomerase activity, anchorage-independent growth and tumor formation in nude mice are the indicators for immortalization, premalignancy and malignancy, respectively.     

亚硝基吡啶对人乳头状瘤病毒诱导的食管上皮永生化细胞的促癌作用

目的 观察亚硝基吡啶在细胞恶性转化过程中的促癌作用.方法 用HPV18E6E7诱导食管上皮细胞永生化细胞系SHEE,第17代细胞培养在50 ml培养瓶.加入亚硝基吡啶（Nnitrosopiperidine,NPIP）0,2,4,8 mmol/L作用3周.用相差显微镜检查细胞形态,流式细胞仪检测细胞增殖和凋亡;染色体常规制样,检查染色体众数;细胞软琼脂集落形成及接种裸小鼠检查成瘤性;用Western blot检测HPV18表达.结果 当细胞暴露在8 mmol/L NPIP时细胞死亡增加,只剩少量活细胞.换正常培基代替NPIP,经4周后细胞进入增殖状态,细胞出现增生和异型增生.第8周末细胞软琼脂培养有大集落形成,接种裸小鼠成瘤.2,4 mmol/L组细胞倍增时间延长,细胞未能成瘤.8 mmol/L NPIP组染色体众数61～65,对照组56～61.实验组和对照组HPV阳性.结论 NPIP促进人乳头状瘤病毒诱导人胚食管永生化上皮恶性转化,HPV18E6E7和NPIP能协同作用加速食管上皮恶性转化.     

Cytogenetic and molecular genetic changes in malignant transformation of immortalized esophageal epithelial cells

The purpose of this study was to evaluate the extent to which the expression of p53, c-myc, bcl-2, ras genes and chromosomes, along with activity of hTERT, impacts on the malignant transformation of immortalized esophageal epithelial cells. The SHEE cell line was established from an embryonic esophageal epithelial cell induced by transduction of E6E7 genes of human papillomavirus type 18 (HPV18E6E7). In cells of the 85th passage (SHEE85), the malignant transformation of SHEE was confirmed by morphology, cell proliferative index and tumor formation in SCID mice. C-myc, p53, bcl-2 and ras genes were assayed by the multi-PCR method with house-keeping gene GAPDH as control. The modal number of chromosomes was analyzed and its expression of subunit of telomerase, hTERT, was assessed by RT-PCR. Expression of HPV18E6E7 was assayed by Western blotting. The results showed that cells of SHEE85 were atypical and exhibited proliferative status with a proliferation index of 45.70%. Tumors formed in SCID mice with invasion of adjacent tissue. The karyotype belonged to hypotriploid and displayed expression of hTERT. C-myc, k-ras, bcl-2 and p53 (expression of phosphoprotein) were positive in SHEE85. Expression of HPV18E6E7 was positive. Taken together, SHEE85 cells were in fully malignant transformation and their molecular mechanism involved the expression of cellular genes, such as p53, bcl-2, c-myc and ras, and aberrance of chromosomes. It is probable that all of these changes were related with HPV18E6E7.     

Immortal phenotype of the esophageal epithelial cells in the process of immortalization

To search for potential biomarkers used to monitor the process of immortalization, we investigated the relative level of telomerase activity and other immortal phenotypes in the SHEE esophageal epithelial cell line. This human fetal esophageal epithelial cell line, induced by human papilloma virus (HPV) 18 E6E7, was continually propagated over 100 passages. Fourteenth passage cells (SHEE14) were cultured in a flask with a serum-free medium and continually cultured to the 30th passage (SHEE30). Cells of SHEE14, SHEE20 and SHEE30 were examined according to cell morphology, cell cycle, apoptosis, contact-inhibition growth, anchorage- dependency, dose-dependency to epithelial growth factors (EGF), telomerase activity and tumorigenicity. The SHEE14 cells exhibited good differentiation with contact-inhibition and anchorage-dependent growth. The SHEE20 cells exhibited increase of senescent and apoptotic cells, and difficulty in propagation. The SHEE30 cells exhibited a higher proliferative index and some undifferentiated cells, with weakened contact-inhibition and anchorage-dependent growth. The telomerase was activated in cells of SHEE30, but not in SHEE14 and SHEE20 cells. The different response to dose-dependency to EGF was not statistically different in SHEE14 and SHEE30. Three groups of cells displayed lack of tumor formation in nude mice. Compared with SHEE14 and SHEE20, SHEE30 cells were of immortalized status with immortal phenotype, which consisted of telomerase activity, increase of cell proliferation, weakened contact-inhibition and anchorage-dependent growth, dose dependency to EGF and lack of tumor formation. From passage 14 to 30th passage, SHEE cells went through cellular senescence, apoptosis and immortalization. With a view toward diagnostic and biological aspects, telomerase activity is a crucial step and a cardinal requirement for immortalization. The telomerase activity and other immortal phenotypes are potential markers for monitoring the process of immortalization.     

丁酸钠对人乳头状瘤病毒诱导的永生化食管上皮细胞恶性转化的促进作用

目的 在人乳头状瘤病毒（HPV）18E6E7基因诱导人胚食管上皮细胞永生化的基础上,观察高、低剂量丁酸钠在细胞恶性转化过程中的促癌作用。方法 永生化食管上皮细胞SHEE先用高剂量于酸钠（80mmol/L),后用低剂量丁酸钠（5mmol/L)各处理8周,再经无丁酸钠条件继续培养14周。用相差显微镜、免疫组织化学SABC法和流式细胞仪检查细胞形态、增殖和调亡状况;用Hoechst33342和碘化丙啶检查活细胞和死细胞;细胞软琼脂集落形成及移植裸小鼠和严重联合免疫缺陷小鼠检查成瘤性。结果 当细胞暴露在80mmol/L丁酸钠,细胞死亡,只剩少量活细胞。在含5mmol/L丁酸钠培养基中细胞出现第一增殖期;撤去丁酸钠,细胞进入危象期,细胞倍增时间延长,如老化细胞。度过危象期,细胞进入第二增殖期,细胞继续增生和异型增生。在第二增殖期末细胞出现恶变,软琼脂培养有大集落形成,移植裸小鼠和SCID小鼠成瘤。结论 SHEE永生化上皮由丁酸钠诱导的恶性变通过了两个阶段的死亡威胁：高浓度丁酸钠引起细胞死亡,缺乏丁酸钠引起细胞危象。高剂量丁酸钠引起永生化细胞死亡,低剂量引起细胞增殖,说明丁酸钠对体外培养细胞有促恶变作用。     

丁酸钠对食管永生化上皮细胞增殖、分化和凋亡的作用

目的 研究丁酸钠对永生化食管上皮的增殖、分化和凋亡的作用。方法 用HPV18E6E7诱发的人胚食管上皮永生化细胞株SHEE,培养在50ml培养瓶和24孔培养板,实验组分别加入1mmol/L和5mmol/L丁酸钠,对照组未加药,作用4d。统计细胞克隆数,细胞超微结构用电镜检查;细胞周期和凋亡细胞数用流式细胞仪检查;Ki-67、细胞角蛋白用免疫组织化学SP法检查;激光共聚焦扫描显微镜检查用鬼臼毒素标记的F-肌动蛋白。结果 加入1mmol/L和5mmol/L丁酸钠4d克隆形成率分别为65.5%和25.5%,比对照组73.5%减少。细胞周期检查1mmol/L组S期细胞明显减少（4.6%),多停留在G0/G1期（83.8%)。与对照组比较,1mmol/L组细胞Ki-67表达降低,F-肌动蛋白和角蛋白表达增加,5mmol/L组细胞凋亡明显增多。结论 丁酸钠可以诱导SHEE细胞增殖停滞和细胞凋亡,并有促细胞分化作用。其与用药剂量和时间有关。     

Identification of pathways and genes in the process of E6/E7-induced carcinogenesis of esophageal epithelial cells

Human papillomavirus (HPV) infection was associated with some carcinomas, especially malignant tumors in upper digestive tract, upper respiratory tract, and genitourinary system. The mechanism of the viral transformation of normal cells is still not very clear. To investigate the tumorigenesis of epithelial cells, E6/E7-induced malignant transformation model cells were used for expression profiling analysis by performing RNA expression microarray detection. Bioinformatics analysis was applied to investigate the cellular process changes along with the E6/E7 expression in SHEE cells. The differentially expressed genes were further grouped and uploaded for Search Tool for the Retrieval of Interacting Genes analysis. The protein-protein interaction results were visualized. The hub genes and their first-neighbors genes were selected, followed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The obtained results demonstrated that tumor-related biological processes began to emerge during the carcinogenesis process from 48th passage to 76th passage of SHEE cells after E6/E7 expression. Ten hub genes were identified and analyzed during the E6/E7-induced tumorigenesis. This study explores the gene expression network in the progressive transformation of immortalized esophageal epithelial cells induced by E6/E7 expression. Understanding the biological processes and hub genes that first appear during the transformation will provide some clues to the mechanism of E6/E7-induced carcinogenesis of esophageal epithelial cells.     

腺病毒伴随病毒表达载体表达人乳头瘤病毒18型E6E7基因构建及其转化作用的鉴定

目的：探讨人乳头瘤病毒（HPV）的E6E7基因在细胞恶性转化中所起的作用。方法：将人乳头瘤病毒（HPV）的E6E7基因克隆至腺病毒伴随病毒表达载体中，通过包装的重组病毒感染，将E6E7基因导入并整合到永生293细胞的基因组中。结果：本研究成功地构建了HPV18 E6E7 AAV病毒并感染了永生293细胞，PCR／Southern杂交分析表明E6E7基因在转化细胞293TL中确有表达，转化细胞293TC和293TL具有明显的转化表型，和亲本293细胞相比，生长速度快，接触抑制消失，集落形成率提高20倍，且集落明显增大，形成时间短。结论：成功地构建了HPV18 E6E7 AAV病毒，HPV18 E6E7基因引起永生化人上皮细胞293的恶性转化。此病毒可用于感染正常上皮细胞，研究其致癌机制。