LNAzyme特异性阻断丙肝病毒5'-NCR/C基因表达

目的 探讨针对HCV 5'-NCR/C双靶基因位点的锁核酸核酶对病毒基因复制与表达的特异性抑制作用.方法 实验分对照组和实验组.对照组分别为空白对照组和脂质体对照组.实验组分别为单靶区NCR组、单靶区C组和双靶区NCR/C组.半乳糖配体介导转染HepG2.9706细胞,用荧光定量PCR检测细胞培养液中HCV mRNA表达;化学发光技术检测细胞培养液中荧光素酶基因表达;荧光显微镜系统检测细胞内荧光蛋白表达;四甲基偶氮唑蓝法检测细胞代谢.应用SPSS 19.0统计学软件分析.各组间比较采用重复测量方差分析的SNK检验和Kruskal Wallis H检验.结果 加入锁核酸核酶后,对HCV RNA的复制均显示有较强的抑制作用(F=77.50,P＜0.05),单靶区NCR组、单靶区C组和双靶区NCR/C组的平均抑制率分别为62.12％、61.39％和75.37％;对荧光素酶的表达有抑制作用(F =48.65,P＜0.05),平均抑制率分别为66.49％、65.06％和73.30％.给药后24、48和96 h,HCV mRNA的平均抑制率分别为52.36％、66.81％和75.05％;荧光素酶的平均抑制率分别为53.02％、62.98％和79.45％;细胞内的荧光蛋白表达阳性细胞数均较对照组明显减少(P＜0.05).结论 针对5'-NCR/C基因位点的LNAzyme能特异性抑制丙型肝炎病毒的复制与表达,且双基因靶位优于单基因靶位.     

HCV基因及其核苷酸序列差异的生物学作用与疾病的关系

近年来，国外关于ＨＣＶ基因组ＲＮＡ序列的研究进展很快。本文就ＨＣＶ的基因分型、核苷酸序列的差异以及ＨＣＶ基因组的多样性与疾病的关系作一简要综述。     

基因中编码区和非编码区的特征序列

为了提取一些能够体现基因序列中编码区和非编码区这两个功能域特征的序列片段--特征序列,本文提出了一个为编码区和非编码区搜索特征序列的新方法.然后应用聚类分析方法验证特征序列提取方法的可靠性.聚类结果表明我们提取的特征序列能够反映基因序列中编码区和非编码区的特性,从而说明提出的提取特征序列的方法是可靠的.     

汉滩病毒S片段基因cDNA3‘端非编码区对核蛋白在…

为了探讨汉滩病毒Ｓ片段ｃＤＮＡ３‘端非编码区基因对核蛋白表达的影响，先后构建了两个核蛋白的表达质粒，其中ｐＢＶＳ２２不含非编码区基因，ｐＢＶＳＸ含有３’端非编码区基因，比较两者在大肠杆菌中表达核蛋白的水平后发现３‘端非码区对核蛋白的表达有明显的负调控作用，表达量下降近５０％，可能的原因是非编码区中含有非常丰富的ＡＴ碱基。该研究结果为进一步提高汉滩病毒核蛋白的表达量积累了经验，也为高效表达其它病毒结     

丙型肝炎病毒基因编码的蛋白及其功能

丙型肝炎病毒(HCV)基因组是一单股正链RNA，全长9600个碱基对(bp)，含有一个大的开放读码框架(ORF)，编码约3010个氨基酸的病毒前体蛋白，HCV基因组5′端(5′UTR)和3′端(3′UTR)为非翻译区，或称非编码区(UCR)，前者位于ORF上游；后者位于下游，含有poly A或ply U尾巴。     

非编码RNA及其调控功能的研究进展

过去认为,在生命活动中RNA只是配角,其作用是将遗传信息从DNA传递给蛋白质,而细胞内存在的小分子RNA（后来称之为非编码RNA,non-coding RNA,ncRNA）只不过是一些降解产物;但随着研究的不断深入,RNA生物学功能的多样性以及ncRNA调控功能的重要性得到了深刻认识。     

卵巢癌中非编码RNA对PTEN基因的调控机制

<正>卵巢癌(ovarian cancer, OC)是女性常见肿瘤,在女性中发病率与死亡率均为第八位、死亡率仅次于宫颈癌^[1],亟需更加有效的治疗方式。OC的发生与PTEN表达水平异常有较强关联;OC有多种组织分型,伴随不同类型的PTEN表达异常,其中,PTEN表达水平下调在子宫内膜样亚型中最常见,     

戊型肝炎病毒摩洛哥株非编码区基因序列鉴定及基因型分型

目的　检测戊型肝炎病毒 (HEV)摩洛哥株非编码区 (UTR)序列 ,探讨HEV非编码区序列能否作为其基因型分型的依据。方法　使用基于RNA连接酶的cDNA末端快速扩增法 (RLM RACE)扩增HEV摩洛哥株 5′和 3′端片段并测序。所得序列使用LASERGENE和PHYLIP软件包与其他 2 9株HEV序列比较。结果　只有基于甲基化帽子结构的RLM RACE扩增出了 5′端片段。HEV摩洛哥株 5′端UTR有 2 6个核苷酸 ,3′端polyA之前有 6 5个核苷酸。基于 3′端UTR序列的进化树与基于全基因序列的进化树不全相同。HEV 3′端至少需要 10 0个左右核苷酸长的序列才可进行HEV基因型分型。结论　HEV摩洛哥株 5′端有甲基化帽子结构。HEV 3′端UTR序列不能完全代替全基因组序列进行基因型分型。部分序列替代全基因组进行基因型分型时需要考虑其部位和长度因素。     

丙型肝炎病毒基因组5''''非编码区结构与功能研究进展

丙型肝炎病毒是经血源传播的一类肝炎病毒,其基因组为单股正链RNA,全长约为9.5kb,结构组成与黄热病毒和瘟病毒相似,它的5非编码区相对较长,一般为324-341个碱基。内含有数个小的开放阅读框。不同分离株在此区的同源性可高达98％,其一级结构高度保守,并可形成特定的二级结构,在病毒基因的表达和复制中起重要的调节作用。     

PAX6基因5′端非编码区剪接突变能够引起先天性无虹膜症

目的对一先天性无虹膜症家系进行了致病基因PAX6的突变分析。方法PCR反应扩增PAX6基因的所有外显子,PCR产物进行SSCP（单链构象多态性）分析,通过患者与正常人带型的差异来确定突变发生的外显子,对有差异SSCP带型的PCR产物进行直接测序找到突变位点。PCR产物进一步亚克隆到pGEM-T载体,测序验证突变位点。结果发现基因突变为PAX6基因第2内含子和第3外显子之间的剪接识别位点＂AG＂中的碱基A的丢失（IVS2-2delA）。结沦PAX6基因5′端非编码区剪接突变能够引起先天性无虹膜症。     

HBV S基因反义锁核酸抑制病毒体外复制

 目的　探讨乙型肝炎病毒S基因反义锁核酸（LNA）片段在HepG22.2.15细胞内抗病毒效果及有效LNA片段筛选。方法　设计合成互补于HBV S基因mRNA翻译起始区同一位点的4条不同序列长度LNA片段,以阳离子脂质体介导,作用于HepG22.2.15细胞株,采用ELISA法和实时荧光定量聚合酶链技术（FQ-PCR）分别监测24、48和72h细胞培养上清液中HBsAg和HBV DNA的含量;四甲基偶氮唑蓝（MTT）法监测细胞代谢。结果　4条不同序列长度反义LNA均显著性抑制HBsAg表达和HBV DNA复制,72h后最高抑制率分别达72.43%和34.73%。HBsAg分泌量和HBV DNA的拷贝数均明显低于对照组（P<0.01）。LNA对细胞代谢无明显影响。结论　针对HBV S基因mRNA翻译起始区的反义LNA片段体外能显著抑制HBV表达,且抑制作用最强序列长度在15~25个碱基之间。     

反基因锁核酸体外抑制乙肝病毒前S1基因表达

 目的 探讨针对乙肝病毒前S1基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的作用。方法 针对乙肝病毒前S1基因同聚嘌呤区,利用RNAstructure软件分别设计合成锁核酸、硫代寡核苷酸、未修饰寡核苷酸及无关对照序列,以阳离子脂质体介导转染HepG2.2.15细胞,采用荧光定量聚合酶链反应技术（FQ-PCR）、时间分辨免疫荧光技术（TRFIA）和酶联免疫法（ELISA）分别监测1、3、5和7 d细胞培养上清液中HBV DNA、HBsAg和前S1抗原的含量;四甲基偶氮唑蓝（MTT）法检测锁核酸对细胞代谢的影响。结果 加入锁核酸后,对HBV DNA复制、HBsAg和前S1抗原表达均显示有较强的抑制作用,且抑制率随时间呈增高趋势,7 d后抑制率分别达64.32%、67.51%和63.88%。LNA对细胞代谢无明显影响。结论 针对乙肝病毒前S1基因同聚嘌呤区的反基因锁核酸,体外能有效抑制乙肝病毒的复制,既为乙肝病毒治疗提供有效靶位,也为反基因治疗提供理论和实验依据。     

Hepatitis C virus (HCV) status in newborns born to HCV positive women performing intracytoplasmic sperm injection

Background

A low risk of HCV vertical transmission has been reported especially in those born to women with viraemia. Intracytoplasmic sperm injection (ICSI) being an assisted reproduction technique includes procedures that may increase risk of transmission.

Objective

To determine the rate of vertical transmission of HCV to newborns born to HCV positive mothers in ICSI cycles.

Methods

In a cross-sectional observational study, serum samples have werecollected within the first week after birth from newborns of two groups of ICSI cycles females. Group one: 30 women HCV antibody (Ab) positive/HCV RNA negative. Group two: 30 women HCV Ab positive/HCV RNA positive. Newborn sera were subjected to HCV antibody testing, and detection of HCV viral RNA by PCR.

Results

None of the newborns born to PCR negative females undergoing ICSI cycles showed positive results neither for HCV antibody, or for HCV RNA. Only one out of the 30 newborns born to PCR positive females was positive for HCV antibody and HCV RNA.

Conclusion

HCV vertical transmission in ICSI cycles seemed to be of low incidence in PCR positive women, while in the case of HCV PCR negative/sero-positive women, it appeared to be completely absent. This observation could have an impact on the clinicians'' counseling for HCV positive females seeking ICSI.     

反基因锁核酸体外阻断肝癌细胞系乙肝病毒S基因表达简

 目的 探讨针对乙肝病毒S基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的效果。方法 针对乙肝病毒S基因同聚嘌呤区设计合成锁核酸、硫代寡核苷酸、未修饰寡核苷酸及无关对照序列,以阳离子脂质体介导,体外转染HepG2.2.15细胞,采用荧光定量聚合酶链反应技术和时间分辨免疫荧光技术分别监测2、4、6、8和10 d细胞培养上清液中HBV DNA、HBsAg和HBeAg的含量;四甲基偶氮唑蓝法检测锁核酸对细胞代谢的影响。 结果 加入锁核酸后,对细胞内HBV DNA复制、HBsAg与HBeAg表达均有较明显的时间和剂量依赖性抑制作用,6 d后抑制率分别为52.14%、57.48%和29.63%。锁核酸对细胞代谢无明显影响。结论 针对乙肝病毒S基因同聚嘌呤区的锁核酸,体外能有效抑制乙肝病毒的复制,既为乙肝病毒治疗提供有效靶位,也为反基因治疗治疗提供理论和实验依据。     

Hepatitis C virus is frequently coinfected with serum marker-negative hepatitis B virus: Probable replication promotion of the former by the latter as demonstrated by in vitro cotransfection

Patients with hepatitis C have been reported occasionally to be coinfected with serum marker-negative (silent) hepatitis B virus (HBV). The frequency and significance of such coinfection were investigated. Thirty patients with hepatitis C virus (HCV) infections (10 acute, 10 chronic, 10 cirrhotic) were selected randomly; the acute cases were without serum hepatitis B surface antigen (HBsAg) and anti-hepatitis B core IgM, and the chronic cases were without HBsAg. A nested polymerase chain reaction for the X open reading frame was used to amplify HBV DNA in serum, and immunoperoxidase staining was carried out on liver biopsy specimens. Nucleotide sequencing was carried out to characterize the amplified HBV DNAs. In order to clarify the possibility that the silent HBV mutant promotes HCV replication in the liver, the full-length HCV RNA and the cloned silent HBV DNA dimer were cotransfected into an established cell line, HuH-7, and the amount of secreted HCV RNA was quantified serially. The target HBV DNA was amplified in 26 (86.7%) of the 30 patients. Subsequent direct nucleotide sequencing in 9 selected patients revealed an 8-nucleotide deletion, characteristic of a silent HBV mutant. Immunostaining revealed hepatitis B surface antigen in 15 (50.0%). Cotransfected silent HBV DNA augmented the secretion of HCV RNA by up to 5-fold in comparison with HCV RNA transfection alone. In conclusion, HCV is coinfected frequently with the silent HBV mutant and the latter probably promotes the replication of the former in the liver. J. Med. Virol. 52:399–405, 1997. © 1997 Wiley-Liss, Inc.     

中国人庚型肝炎病毒E1区5＇端基因相关多肽的抗原性初步研究

用逆转录－ＤＮＡ聚合酶链式反应（ＲＴ－ＰＣＲ）从１例中国庚型肝炎（ＨＧＶ）患者血清中扩增出含Ｅ１前区部分基因及Ｅ１区５＇端共３０９ｈｐ基因片段。对其中包括Ｅ１区９６ｂｐ在内的１２０ｂｐ核苷酸进行了序列分析，发现此区段基因与录入号为Ｕ４４４０２Ｇｅｎｅｂａｎｋ报道的美国发现的ＨＧＶ相关序列具有９３％的同源性，氨基酸的同源性为９８％。Ｇｏｌｄｋｅｙ蛋白质分析程序显示，此区段的Ｅ１区可能存在抗原位点。化学合成可能为抗原位点长约２９个氨基酸的多肽Ｅ１Ｐ１。利用Ｅ１Ｐ１及ＮＳ３区内短肽ＮＳ３Ｐ１包被的ＥＬＩＳＡ方法检测了１２名非甲－戊肝炎患者血清。Ｅ１Ｐ１检出的２份血清（２／１２）中有抗－Ｅ１Ｐ１ＩｇＧ的存在。ＮＳ３Ｐ１捡出的３份血清（３／１２）中有抗－ＮＳ３Ｐ１ＩｇＧ的存在，其中包括Ｅ１Ｐ１检出的两份阳性血清。本结果说明在ＨＧＶＥ１区内至少有一个抗原位点存在。     

Hepatitis B virus (HBV) reactivation—The potential role of direct‐acting agents for hepatitis C virus (HCV)

Hepatitis C virus (HCV) is known to inhibit hepatitis B virus (HBV) replication in patients with HBV/HCV coinfection. Reactivation of HBV in patients treated for HCV with direct‐acting agents (DAAs) has emerged recently as an important clinical consideration. A growing number of case reports and case series support the association between new HCV treatments and HBV reactivation. Yet, very little is known about the specific viral characteristics that facilitate reactivation as functional characterization of the reactivated HBV has been conducted only rarely. This review provides the most recent data on HBV reactivation in the context of DAA initiation and highlights the existing viral genomic data from reactivating viruses. Current functional studies of HBV reactivation are largely limited by the retrospective identification of cases, no standardization of genomic regions that are studied with respect to HBV reactivation, and the lack of inclusion of nonreactivating controls to establish specific viral mutations that are associated with HBV reactivation. Importantly, none of these sequencing studies included cases of HBV reactivation after initiation of DAAs. While new HCV treatments have revolutionized care for HCV infected patients, HBV reactivation will likely increase in frequency, as DAAs are more commonly prescribed. Pretreatment determination of HBV status and thoughtful management of HBV coinfections will be necessary and lead to improved patient safety and yield optimal treatment results.     

Quasispecies nature of hepatitis C virus (HCV) in patients with chronic hepatitis C with mixed HCV subtypes

The quasispecies nature of hepatitis C virus (HCV) in patients with mixed HCV subtype infection was compared with that in patients with single HCV subtype infection. The number of HCV quasispecies was compared between 35 patients with mixed HCV subtype infection and 83 patients with single subtype infection. Subtype was determined by primers deduced from the core region and by line probe assay respectively. The number of quasispecies was evaluated by polymerase chain reaction amplification of hypervariable region 1 and by fluorescence single-strand conformation polymorphism analysis. There was no difference in clinical background between patients with mixed subtype infection and patients with single subtype infection. The number of quasispecies in patients with multiple subtype HCV infection was larger than in patients with single subtype HCV infection. The immunologic environments which allow the coexistence of more HCV quasispecies in patients with multiple HCV subtype infection differs from that in patients with single HCV subtype infection. J. Med. Virol. 54:80–85, 1998. © 1998 Wiley-Liss,Inc.