端粒酶测定在肿瘤诊断中的应用

一、端粒 (telomere)端粒是位于真核细胞染色体末端 ,由 DNA和蛋白质组成的特殊结构 ,DNA内含有大量(TTAGGG) n的重复序列。DNA复制沿 5′→ 3′方向进行 ,由 RNA引物起始聚合反应。 DNA合成结束后 RNA引物降解 ,DNA聚合酶有填补缺口的作用 ,但是与模板DNA3′端结合的引物降解后不能被填补 ,因此在DNA复制过程中染色体末端有一段遗传信息得不到复制。随着细胞不断分裂 ,DNA3′端的序列也就不断丢失 ,这就是所谓“末端复制问题”。由于 DNA复制过程中存在“末端复制问题”,在细胞分裂中 ,端粒的长度会逐渐缩短。当端粒缩短到…     

血管生成相关miRNAs与子痫前期

MiRNAs是一类长度约9~25个核苷酸的非编码单链小分子RNA,由60~110 nt的内源性转录前体经过Drosha酶和Dicer酶加工而成。Mi RNA通过与目标m RNA分子的3′端非编码区(3′-untranslated region,3′UTR)的完全或不完全配对引起靶m RNA的降解或抑制其翻译,从而对基因进行转录后表达的调控[1]。Mi RNA对靶基因的转录后调节在血管再生及内皮细胞功能调控中起重要作用,如miR-126能在内皮细胞中特异性表达并调控血管生成;miR-210在缺氧导致的血管生成中发挥重要作用;miR-17/92簇     

扇贝防御素基因改良5′cDNA末端快速扩增聚合酶链反应筛选方法的研究

目的应用改良5′cDNA末端快速扩增聚合酶链反应(RACE)筛选扇贝抗菌肽基因,寻找基于基因序列同源性的双壳类抗菌肽(AMP)的筛选方法。方法 TRIzol法提取扇贝血淋巴中总RNA。根据贻贝抗菌肽的保守序列设计基因特异性简并引物,进行改良5′RACE钓取可能防御素基因cDNA的5′端序列、AT克隆、DNA测序和同源性分析。结果采用改良5′RACE从扇贝血淋巴RNA中得到多个未知基因的cDNA 5′端序列,挑选600bp以下的基因片段进行AT克隆和DNA测序后得到3个插入片段序列,其长度分别为257、275和511bp。通过BLAST程序搜索GenBank核酸数据库,未发现与之高度同源的基因。结论从扇贝血淋巴中获得的3个5′端cDNA序列可能属于新的基因。应用改良5′RACE能钓取含防御素保守序列的新基因片段,由此表明此方案具有可行性。     

端粒酶抑制剂的研究进展

端粒是真核细胞染色体末端的特有结构，正常情况下随细胞分裂而缩短。端粒长度是决定细胞增殖能力和寿命的分子标志。端粒酶是存在于大多数恶性肿瘤细胞中的一种转录酶。它能以自身的RNA成分为模板，合成端粒染色体末端的结构，维持染色体的动态平衡。端粒酶是目前最为广谱的肿瘤分子标志物。针对端粒酶的抑制剂成为肿瘤治疗的新靶点。肿瘤治疗的理想靶点是既能筛选出肿瘤生长所必须，而在正常细胞中又不存在的物质或表达状态。目前针对抑制端粒酶活性的肿瘤治疗研究已经取得了很多成果，并有可能直接导致肿瘤治疗的突破。     

端粒酶逆转录酶的研究进展

端粒是真核细胞染色体末端的特有结构，正常情况下随细胞分裂而缩短。端粒长度是决定细胞增殖能力和寿命的分子标志。人端粒酶是一种核糖核蛋白复合物，由人端粒酶逆转录酶（hTERT）、人端粒酶RNA组分（hTR）以及人端粒酶相关蛋白（hTEPl等）组成。端粒酶利用其自身hTR所携带的RNA为模板，在hTERT的逆转录催化下，将端粒重复序列合成到染色体末端，     

NASBA和IS-NASBA的临床应用及研究进展

核酸序列依赖扩增(NASBA)是一种特异性等温扩增RNA的技术,是基于一个等温扩增酶系统,包括AMV逆转录酶(AMVRT)、RNaseH和T7RNA聚合酶。以RNA为模板,在AMV逆转录酶和引物Ⅰ的作用下合成cDNA,引物Ⅰ的5′端带有T7启动子序列,然后引物Ⅱ与cDNA链退火合成cDNA链的互补链,进而形成了含完整T7启动子序列的双链DNA。T7RNA聚合酶以此双链DNA为模扳,合成大量拷贝的RNA片段。原位NASBA(insituNASBA,ISNASBA)是结合原位杂交技术而发展起来的,主要用于分子病理学、分子形态学领域的杂交技术。     

circRNA与肿瘤发生的研究进展

环状RNA(Circular RNA,circRNA)是近年来发现的一种新的内源性非编码RNA。circRNA不具有5'末端帽子和3'末端多聚A尾,呈现闭合环状结构,这种特殊结构使circRNA具有高度的保守性和稳定性。circRNA主要由外显子形成,大量存在于真核细胞中。circRNA比线性的mRNA含有更丰富的转录本,能够在转录或转录后水平调控多种生命活动。而且,circRNA还可作为竞争性内源RNA(ceRNA)的组成部分,抑制miRNA的活性,从而调控基因转录、翻译等功能。作为一种新型调控分子及研究热点,circRNA在肿瘤等多种疾病中异常表达,有望成为新的诊断及预测肿瘤发生发展的生物标志物。     

1株人感染柯萨奇病毒A组16型B1b亚型的分离和鉴定

正柯萨奇病毒A16(CV-A16)是小RNA病毒科肠道病毒属的成员,是引起婴幼儿手足口病(HFMD)的常见病原体之一[1]。CV-A16是含有约7.4kb基因组的正义单链RNA病毒,其基因组包含5′端非翻译区(5′UTR)、结构蛋白编码区P1、非结构蛋白编码区P2/P3及3′非翻译区(3′UTR)。P1编码4种结构蛋白(VP1、VP2、VP3、VP4),P2和P3分别编码7种非结构蛋白(2A、2B、2C、3A、3B、3C、3D)[2]。随着研究的不断深入,在分子流行病学方面,基于VP1基因的完整序列,     

HLA-DRB1-12寡核苷酸芯片的制备及优化

生物芯片技术是基于杂交原理发展而来的，是将固相反应的原理和形式应用于大分子识别反应(核酸杂交、抗原抗体结合或酶促的模板依赖性的连接、延伸等反应)中，以达到对大量的目标分子进行快速、平行的特异识别。寡核苷酸芯片的制备过程中关键部分是基片的表面化学处理和探针末端的不同修饰。为了比较不同末端修饰探针在不同化学处理的载玻片上的杂交信号的强弱，本研究根据HLA—DRBl—12的序列设计8种不同类型的探针，即4种5′末端修饰探针，包括末端氨基修饰探针(N)，氨基加四聚乙二醇间隔臂探针(NL)，硫代探针(S)，硫代加四聚乙二醇间隔臂探针(SL)和4种3′末端修饰探针，同样包括末端氨基修饰探针，氨基加四聚乙二醇间隔臂探针，硫代探针，硫代加四聚乙二醇间隔臂探针。将这8种探针分别固定在溴化芯片和醛基芯片上。与末端标记荧光的不对称的PCR产物进行杂交，通过比较杂交结果荧光信号的强弱，筛选出探针同活化基片的最佳组合。从而达到优化寡核苷酸芯片制备的目的。另外，为了进一步比较四聚乙二醇间隔臂对杂交信号的影响。设计末端连接不同数目四聚乙二醇的3′氨基探针。结果显示，3′末端修饰探针杂交信号强于5′末端修饰探针，探针在溴化芯片中的杂交信号强度高于在醛基芯片中杂交信号，3′带有间隔臂的R氨基探针在溴化芯片中的杂交信号最强。另外3′氨基探针中间隔臂可以增强杂交信号强度，但随着数目的增加，杂交信号并不显增加。结论：溴化芯片中3′末端带间隔臂的氨基修饰探针在杂交过程中可以更有效的捕获靶标，提高寡核苷酸芯片的杂交能力；四聚乙二醇的数量不必过多使用；通过这种方法可以达到优化芯片制备的目的，为下一步HLA寡核苷酸分型芯片及其他基因芯片的研制提供有效的手段。     

miR-361在心血管疾病的研究进展

微小RNA(microRNA,miRNA)是一类内源性单链非编码RNA分子,长度约21～23个核苷酸,通过结合靶基因的3′非翻译区转录调控基因的表达,参与细胞增殖、分化、凋亡等生物学活动,对机体内不同组织细胞的生长、发育产生重要影响.随着近几年对miRNA-361的深入研究,发现miRNA-361在心血管方面具有重要作...     

长链非编码RNA在肿瘤发生及发展中的作用机制

长链非编码RNA(lncRNA)是一类转录本长度＞200个核苷酸的非编码RNA(ncRNA),其在肿瘤的发生、发展中发挥重要作用.IncRNA具有多种生物学功能,可以通过诱饵分子、支架分子和向导分子等形式,在表观遗传学水平、转录水平及转录后水平,调控蛋白质编码基因的表达.lncRNA与肿瘤细胞生长和凋亡、侵袭和转移等生物学行为密切相关.进一步阐明IncRNA在肿瘤发生、发展过程中的作用机制,有望为研究者开发肿瘤诊断和治疗的新方法提供思路.笔者拟就lncRNA调控基因表达的作用机制,及其在肿瘤诊断及治疗中的作用进行综述.     

The role of small RNAs in human diseases: potential troublemaker and therapeutic tools

Small RNAs, including short interfering RNAs (siRNAs) and microRNAs (miRNAs), are ubiquitous, versatile repressors of gene expression in plants, animals, and many fungi. They can trigger destruction of homologous mRNA or inhibition of cognate mRNA translation and play an important role in maintaining the stable state of chromosome structure and regulating the expression of protein-coding genes. Furthermore, the recent research showed that there exists close relationship between small RNAs and human diseases. Several human diseases have surfaced in which miRNAs or their machinery might be implicated, such as some neurological diseases and cancers. The specificity and potency of small RNAs suggest that they might be promising as therapeutic agents. This article will review the role of small RNAs in some human diseases etiology and investigations of taking siRNAs as therapeutic tools for treating viral infection, cancer, and other diseases. We also discuss the potential of miRNAs in gene therapy.     

长链非编码RNA与肿瘤诊断

非编码RNA参与了多种疾病尤其是肿瘤发生发展的调控过程,是近期研究热点之一。随着高通量筛选方法的完善,越来越多的lncRNA分子被发现,并有望成为新型肿瘤诊断标志物和肿瘤治疗的靶点。近期研究提示IncRNA在肿瘤诊断和治疗方面具有良好的临床应用前景。本文介绍了lncRNA近期研究进展,相关lncRNA数据库的使用,并着重介绍了lncRNA与肿瘤诊断和预后关系研究情况。     

Advances in microRNAs: implications for gene therapists

MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to regulate the expression of as many as one-third of all human messenger RNAs (mRNAs). miRNAs are thought to be involved in diverse biological processes, including tumorigenesis. Analysis of miRNA levels may have diagnostic implications. Evidence shows that numerous viruses interact with the miRNA machinery, and that a number of viruses encode their own miRNAs. It seems likely that miRNAs will be implicated in many human diseases. Manipulation of miRNA levels by gene therapy provides an attractive new approach for therapeutic development. This review focuses on approaches to manipulate miRNA levels in cells and in vivo, and the implications for gene therapy. Furthermore, we discuss the use of endogenous miRNAs as scaffolds for the expression of RNA interference (RNAi) as well as competition between exogenous RNAi triggers and endogenous miRNAs. Because short interfering RNAs can also act as miRNAs, seed matches with the 3' untranslated regions of genes should be avoided to prevent off-target effects. Last, we discuss the use of miRNAs to avoid immune responses to viral vectors.     

Role of non-coding RNAs in pathogenesis of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are considered the model solid malignancies of targeted therapy after the discovery of imatinib effectiveness against their tyrosine kinase inhibitors. Non-coding RNAs are molecules with no protein coding capacity that play crucial role to several biological steps of normal cell proliferation and differentiation. When the expression of these molecules found to be altered it seems that they affect the process of carcinogenesis in multiple ways, such as proliferation, apoptosis, differentiation, metastasis, and drug resistance. This review aims to provide an overview of the latest research papers and summarize the current evidence about the role of non-coding RNAs in pathogenesis of GISTs, including their potential clinical applications.     

Combinatorial RNA-based gene therapy for the treatment of HIV/AIDS

Introduction: HIV/AIDS continues to be a worldwide health problem and viral eradication has been an elusive goal. HIV+ patients are currently treated with combination antiretroviral therapy (cART) which is not curative. For many patients, cART is inaccessible, intolerable or unaffordable. Therefore, a new class of therapeutics for HIV is required to overcome these limitations. Cell and gene therapy for HIV has been proposed as a way to provide a functional cure for HIV in the form of a virus/infection resistant immune system.

Areas covered: In this review, the authors describe the standard therapy for HIV/AIDS, its limitations, current areas of investigation and the potential of hematopoietic stem cells modified with anti-HIV RNAs as a means to affect a functional cure for HIV.

Expert opinion: Cell and gene therapy for HIV/AIDS is a promising alternative to antiviral drug therapy and may provide a functional cure. In order to show clinical benefit, multiple mechanisms of inhibition of HIV entry and lifecycle are likely to be required. Among the most promising antiviral strategies is the use of transgenic RNA molecules that provide protection from HIV infection. When these molecules are delivered as gene-modified hematopoietic stem and progenitor cells, long-term repopulation of the patient's immune system with gene-modified progeny has been observed.     

Interference between effector RNAs expressed from conventional dual-function anti-HIV retroviral vectors can be circumvented using dual-effector-cassette retroviral vectors

Coexpression of different effector molecules from a single vector (a dual-function vector) may provide enhanced efficacy. Thus far most of the reported anti-HIV dual-function vectors express different effector RNAs as a chimeric molecule. In our study involving retroviral vectors coexpressing a U5 ribozyme and either an anti-tat or anti-rev antisense RNA, chimeric vectors exhibit poor potency in several important functional aspects, including inhibition of HIV replication, protection against cytopathic effects, and suppression of target gene function. Surprisingly, such a poor efficacy of chimeric vector function was not associated with a lower level of effector RNA expression. These results indicate that expression of two effector RNAs as a chimeric molecule can lead to interference, reducing their global biological effects. More importantly, we have demonstrated that such interference can be avoided by coexpressing these effector RNAs as separate molecules through a new dual-function vector, called a dual-effector cassette (Dec) vector, developed in this study. We also define some of the design alterations that might affect the efficacy of the Dec vector and demonstrate that forward-designed Dec vectors are more efficacious than reverse-designed Dec vectors, which express a lower level of effector RNA owing to the instability of the 5' effector cassettes in the provirus. We believe that the principle of Dec vector design may also be applicable for the coexpression of other therapeutic RNA effectors in many gene therapy applications.