植酸酮对宫颈癌细胞株中 HPV16／18 E6／E7 mRNA 及蛋白表达的影响

目的观察植酸酮对宫颈癌细胞株中HPV16/18 E6/E7 mRNA及蛋白表达的影响。方法体外培养Caski细胞株(含HPV16)与Hela细胞株(含HPV18),将所有细胞分成实验1、2、3、4组和对照组,每组均包含Caski细胞和Hela细胞。实验1、2、3、4组分别加入含58.6、117、586、5 860 mg/L植酸酮的培养液,对照组加入不含植酸酮的培养液。各组培养72 h后,采用real-time PCR法分别检测HPV16/18 E6/E7 mRNA,采用Western blotting法检测E6/E7蛋白。结果实验1、2、3、4组同一型别细胞中E6/E7 mRNA及蛋白相对表达量低于对照组,且实验1、2、3、4组E6/E7 mRNA及蛋白相对表达量依次降低(P均<0.05)。各实验组内,Caski细胞中E6/E7 mRNA相对表达量低于Hela细胞(P均<0.05)。各实验组内同一型别细胞中,E6 mRNA相对表达量低于E7 mRNA(P均<0.05)。结论植酸酮可下调人宫颈癌细胞株中HPV16/18 E6/E7 mRNA及蛋白表达,且作用呈剂量依赖性;植酸酮对HPV16 E6/E7 mRNA的抑制作用强于HPV18 E6/E7 mRNA,并可能以抑制E6 mRNA表达为主导。     

HPV16 E7 siRNA表达载体对宫颈癌CaSki细胞生物学活性的影响

目的 探讨人乳头瘤病毒16型(HPV16) E7基因小干扰RNA(siRNA)表达载体对宫颈癌CaSki细胞生物学活性的影响.方法 利用脂质体HPV16 E7特异性siRNA表达载体(HPV16-E7Si)、空载体(CaSki-Si)转染CaSki细胞,荧光定量RT-PCR、流式细胞仪检测HPV16-E7Si、CaSki-Si细胞E7 mRNA和蛋白的变化;流式细胞仪及MTT法检测2种细胞的细胞周期及生长曲线变化,并检测2种细胞对顺铂的敏感性.结果 成功获得沉默E7基因的克隆细胞株CaSki-E7Si及阴性对照CaSki-Si;CaSki-E7Si细胞生长明显慢于CaSki-Si细胞(P＜0.05),且前者细胞的凋亡峰明显增多.2种细胞加用顺铂作用48 h后发现,CaSki-Si细胞周期阻滞在S～ G2期,HPV16-E7Si阻滞在G0～1期.结论 HPV16 E7 siRNA表达载体能有效地抑制宫颈癌CaSki细胞E7基因的表达,促进肿瘤细胞凋亡,并增加顺铂的敏感性.     

雌激素通过其受体α上调LRP16 mRNA表达并促进MCF-7细胞增殖

目的 探讨雌激素对人乳腺癌MCF-7细胞LRP16 mRNA表达的调控作用以及过表达LRP16对MCF一7细胞生长特性的影响。方法 (1)用Northem印迹方法检测17β-雌二醇(17β-E2)对LRP16m RNA表达水平的作用；(2)构建LRP16启动子(-2．6kb)调控的荧光素酶报告子(pG13-S0)，并与各种核受体包括雌激素受体α和β(ERα和ERβ)、糖皮质激素受体α(GRα)、雄激素受体(AR)和过氧化物酶体增殖物激活的受体γ和α(PPARγ和PPARα)表达载体共转染COS-7细胞，测定荧光素酶活性；(3)将LRP16表达载体转染MCF-7细胞，测定过表达LRP16对细胞的生长特性和细胞周期的影响，并用Western印迹方法测定周期素E、p53和p21^WAF1/CIP1蛋白的变化。结果 (1)17β-E2使MCF-7细胞的LRP16 mRNA表达水平增加5～8倍，增加幅度未显示出β-E2培养时间和剂量的依赖性；(2)pGL3-S0与ERα或AR共转染细胞的相对荧光素酶活性较单独转染pGL3-S0细胞(对照组)分别升高11和7．8倍；(3)LRP16过表达促进MCF-7细胞的生长且伴有周期素E显著升高，S期细胞的数量较对照组增加约10％。结论 (1)雌激素通过激活ERα增加MCF-7细胞LRP16 mRNA的表达；(2)过表达LRP16可能通过上调周期素E促进MCF-7细胞生长。     

老年宫颈癌组织中人乳头状瘤病毒16-E6、p53 mRNA表达及其与放疗敏感性的相关性

目的分析老年宫颈癌组织中人乳头状瘤病毒(HPV)16-E6、p53 mRNA表达及其与放疗敏感性的关系。方法 40例宫颈癌患者均按计划进行根治性放射治疗前后采用实时定量反转录聚合酶链反应(PT-PCR)检测宫颈癌组织中HPV16-E6、p53 mRNA的表达水平,并根据治疗结果分为有效组29例和无效组11例,分析HPV16-E6、p53与放疗敏感性的关系。结果治疗总有效率为72.50%;放疗前后无效组2例HPV16-E6、p53 mRNA和蛋白差异无统计学意义(P>0.05),放疗前后有效组4例HPV16-E6 mRNA、p53 mRNA表达水平明显改善,HPV16-E6蛋白表达水平显著低于无效组,p53蛋白表达水平显著高于无效组(P<0.05)。结论 HPV16-E6感染、p53基因异常参与了老年宫颈癌发生、发展过程,检测HPV16-E6、p53 mRNA的表达可判断宫颈癌组织放疗敏感性。     

RNAi对人乳腺癌MCF-7细胞E2F1、TS基因表达的抑制作用

目的观察RNA干扰（RNAi）技术对乳腺癌细胞MCF-7中E2F1、胸苷酸合成酶（TS）基因表达的靶向抑制作用。方法设计合成并构建重组质粒pSIREN-E2F1，将重组质粒转染MCF-7细胞。筛选稳定转染的单克隆细胞扩大培养，应用RT-PCR方法检测各组细胞E2F1、TS mRNA表达情况。结果转染组E2F1、TS基因表达均受到抑制：结论RNAi技术特异、高效抑制靶基因E2F1及TS表达。     

Cenicriviroc对HeLa细胞CCL2/CCL5 APOBEC3A/B以及HPV18 E6/E7 mRNA转录水平的影响

目的观察cenicriviroc处理后HeLa细胞中CCL2/CCL5、载脂蛋白B信使核糖核酸(mRNA)编辑酶催化多肽样蛋白3(APOBEC3)A/APOBEC3B(A3A/A3B)以及人乳头瘤病毒(HPV)18早期基因E6/E7 mRNA的相对表达量,探讨趋化因子受体与APOBEC3,以及与HPV早期基因mRNA转录水平间的关联性。方法 MTT法检测不同浓度cenicriviroc对HeLa细胞的毒性;实时荧光定量反转录聚合酶链反应(qRT-PCR)检测50和25 nM cenicriviroc作用于HeLa细胞12、24、48 h后胞中CCR2/CCR5、CCL2/CCL5、A3A/A3B和HPV18 E6/E7 mRNA的相对表达量,并与未处理的HeLa细胞作比较。结果 100 nM以下的cenicriviroc作用48 h内对HeLa细胞基本无毒性。50 nM cenicriviroc在处理12、24、48 h后,对CCR2/CCR5、CCL2/CCL5和HPV18 E6/E7 mRNA的转录水平均有一定的下调作用;而25 nM组仅在处理12 h时能显著下调CCR2、E6 mRNA的转录水平(P0.05)。除12 h 25 nM组与对照组间A3A基因mRNA的相对表达量差异无统计学意义外,其余各时间点50、25 nM组均可明显升高A3A mRNA的相对表达量(P0.05);两个浓度组胞内A3B mRNA的相对表达量均没有明显变化(P0.05)。结论 cenicriviroc在体外能抑制HeLa细胞CCR2/CCR5、CCL2/CCL5、HPV18 E6/E7 mRNA的转录水平,并上调胞内A3A mRNA的转录。     

宫颈脱落细胞HPV E6/E7mRNA、HR-HPV DNA在宫颈病变筛查中的应用

目的 探讨HPV E6/E7 mRNA与高危型孔头瘤病毒（HR-HPV） DNA在宫颈病变筛查中的应用价值.方法 对301例宫颈病变疑似病例进行宫颈脱落细胞取样,分别检测慢性宫颈炎、宫颈癌前病变、宫颈鳞状细胞癌中HPV E6/E7 mRNA与HR-HPV DNA的表达,比较二者在不同年龄组及病变程度中表达的差异.结果 在宫颈癌前病变及宫颈鳞状细胞癌患者中HPV E6/E7 mRNA的特异性和阳性预测值高于HR-HPV DNA（P均＜0.05）,敏感性低于HR-HPV DNA（P ＜0.05）.30岁以下与30～39岁受检者中HPV E6/E7 mRNA的阳性率低于HR-HPV DNA（P均＜0.05）.CIN2、CIN3及宫颈癌患者HPV E6/E7 mRNA阳性率高于慢性宫颈炎患者,且CIN3者高于CIN2者（P均＜0.05）,CIN1、CIN2、CIN3及宫颈癌患者HR-HPV DNA阳性率高于慢性宫颈炎患者（P均＜0.05）.结论 对于宫颈癌及癌前病变,HPV E6/E7 mRNA的阳性率及其诊断的特异性高于HR-HPV DNA,可作为筛查和随访的指标.     

HPV16/18E6和P53基因蛋白在喉鳞癌中的表达及其相关性研究

目的探讨人乳头瘤病毒16/18型早期蛋白E6(HPV16/18 E6)、P^53基因蛋白在喉鳞癌(LSCC)中的表达及其相关性.方法免疫组化SABC法检测HPV16/18E6和P^53基因蛋白在LSCC及声带息肉中的表达.结果 HPV16/18E6在声带息肉中未见表达,在LSCC中的表达率为40.0%,差异有显著性(P＜0.05). P^53基因蛋白在声带息肉与LSCC中的表达率分别为6.66%和66.2%,差异有显著性(P＜0.05).HPV16/18E6及 P^53基因蛋白的表达与LSCC临床分期、淋巴结转移有关.二者的表达无明显相关性.结论 HPV16/18型感染和P^53基因异常与LSCC发生、发展有关.HPV16/18E6与P^53基因功能异常无明显相关性.     

弓形虫ROP16蛋白对人乳腺癌MCF-7细胞增殖、周期和凋亡的影响

目的 探讨弓形虫Ⅰ型(RH)ROP16蛋白在人乳腺癌MCF-7细胞增殖、周期及凋亡方面的作用。方法 以空载体(MCF-7-HBLV)和过表达ROP16蛋白的慢病毒(HBLV-RH ROP16)分别感染MCF-7细胞,嘌呤霉素筛选出稳定表达ROP16的细胞株,Real time PCR、Western blot法检测MCF-7细胞中ROP16 mRNA和蛋白表达水平。CCK-8和流式细胞术检测细胞增殖、周期和凋亡。Western blot法检测细胞周期及凋亡相关蛋白表达。结果 相比MCF-7-HBLV(空载体组)和MCF-7细胞组(对照组),MCF-7-RH ROP16细胞组中ROP16 mRNA和蛋白表达升高;细胞增殖率降低(P<0.01);S期细胞比例、细胞凋亡率升高(P<0.01)。促凋亡因子Bax、P53、Caspase-9、Caspase-3及细胞周期蛋白依赖激酶抑制因子P21表达增高(P<0.01);抗凋亡蛋白Bcl-2及细胞周期蛋白A1(CyclinA1)、周期蛋白依赖性激酶2(CDK2)的表达均下降(P<0.01)。结论 弓形虫Ⅰ型(RH)ROP1...     

人乳头状瘤病毒16型E6E7基因与MCA、TPA协同诱导细胞恶性转化的实验研究

目的探讨人乳头状瘤病毒(HPV)16型E6E7基因与化学致癌物MCA、TPA对Balb/c 3T3细胞恶性转化的协同作用。方法构建含HPV16 E6E7基因的重组质粒,用其转染Balb/c 3T3细胞。采用RT-PCR和Westernblot技术检测HPV16 E6E7基因和蛋白表达;应用细胞转化实验研究由MCA和TPA诱导的细胞恶性转化;并检验转化细胞在软琼脂上形成集落的能力及对SCID小鼠的致瘤能力。结果转染HPV16 E6E7基因的细胞比未转染细胞形成更多的转化灶,转化灶个数增加4～25倍,且实验时间明显缩短;其转化细胞在软琼脂上形成集落的能力及对SCID小鼠的致瘤能力更强。结论 HPV16 E6E7基因与MCA、TPA可协同诱导Balb/c 3T3细胞恶性转化。     

Hepatitis E

Hepatitis E, previously known as enterically transmitted non-A, non-B hepatitis, is an infectious viral disease with clinical and morphologic features of acute hepatitis. Its causative agent, hepatitis E virus, consists of small, 32- to 34-nm diameter, icosahedral, nonenveloped particles with a single-stranded, positive-sense, 7.5-kb RNA. The virus has two main geographically distinct strains, Asian and Mexican; recently, novel isolates from nonendemic areas and a genetically related swine HEV have been described. HEV is responsible for large epidemics of acute hepatitis and a proportion of sporadic hepatitis cases in the Indian subcontinent, southeast and central Asia, the Middle East, parts of Africa, and Mexico. The virus is excreted in feces and is transmitted predominantly by fecal-oral route, usually through contaminated water. Person-to-person transmission is uncommon. Clinical attack rates are the highest among young adults. Recent evidence suggests that humans with subclinical HEV infection and animals may represent reservoirs of HEV; however, further data are needed. Diagnosis of hepatitis E is usually made by detection of specific IgM antibody, which disappears rapidly over a few months; IgG anti-HEV persists for at least a few years. Clinical illness is similar to other forms of acute viral hepatitis except in pregnant women, in whom illness is particularly severe with a high mortality rate. Subclinical and unapparent infections may occur; however, chronic infection is unknown. No specific treatment is yet available. Use of clean drinking water and proper sanitation is currently the most effective method of prevention. Passive immunization has not been proved to be effective, and recombinant vaccines for travelers to disease-endemic areas and for pregnant women currently are being developed.     

Hepatitis E

Hepatitis E refers to liver disease caused by the hepatitis E virus (HEV), a small, nonenveloped virus with a single-stranded RNA genome. The virus has four genotypes, but only one serotype. Genotypes 1 and 2 exclusively infect humans, whereas genotypes 3 and 4 also infect pigs and several other mammalian species. Though HEV does not grow well in cell culture, several aspects of its biology and pathogenesis have been worked out using animal models and cell transfection studies, and by analogy with other related viruses. HEV itself appears noncytopathic, and the liver injury during hepatitis E may be mediated by the host immune response. In areas with poor sanitation, HEV infection is common and presents as outbreaks and also as sporadic cases with acute self-limited hepatitis. The transmission is feco-oral, usually through contaminated drinking water. The disease often affects young adults and is particularly severe among pregnant women and persons with preexisting liver cirrhosis. In the developed world, the disease is being increasingly recognized. It occurs as occasional sporadic cases, most often among elderly men with coexisting illnesses. These appear to be related to zoonotic transmission. Chronic infection is known among immunosuppressed persons in these regions and may progress to liver cirrhosis. Serological tests for diagnosis of HEV exposure and recent infection, namely immunoglobulin (Ig)G and IgM anti-HEV, respectively, need further improvement in sensitivity and specificity, particularly when used in developed countries. Two recombinant protein vaccines have undergone successful human trials, but are not yet commercially available. Recent development of cell-culture methods for HEV should allow a better understanding of this enigmatic agent.     

Hepatitis E

Hepatitis E virus (HEV) was discovered during the Soviet occupation of Afghanistan in the 1980s, after an outbreak of unexplained hepatitis at a military camp. A pooled faecal extract from affected soldiers was ingested by a member of the research team. He became sick, and the new virus (named HEV), was detected in his stool by electron microscopy. Subsequently, endemic HEV has been identified in many resource-poor countries. Globally, HEV is the most common cause of acute viral hepatitis. The virus was not initially thought to occur in developed countries, but recent reports have shown this notion to be mistaken. The aim of this Seminar is to describe recent discoveries regarding HEV, and how they have changed our understanding of its effect on human health worldwide.     

Hepatitis E

Hepatitis E has a world-wide distribution and causes substantial morbidity and mortality in some developing countries, particularly among pregnant women. Hepatitis E virus (HEV) has recently been cloned and sequenced, and new diagnostic tests have been developed. These tests have been used to begin to characterize the natural history and epidemiological features of HEV infection. Experimental vaccines have also been developed that offer the potential to prevent hepatitis E. However, much remains to be learned about HEV, including the mechanisms of transmission, the reservoir(s) of the virus, and the natural history of protective immunity in order to develop effective strategies to prevent this disease.