RNA干扰引起的非特异性免疫反应及其机制研究探讨

基因的表达失控是疾病发生的主要原因之一,干扰靶基因的表达可能成为有效的治疗手段。RNA干扰技术是近年兴起的基因调控干预方法,其基础,特别是应用研究极受关注,人们期待RNA干扰能成为肿瘤、病毒感染等难治疾病的临床治疗手段。然而,这一新兴技术在应用研究过程中显现出诸多问题,如细胞毒性、引起机体非特异性反应等等。就RNA干扰引起的非特异性免疫反应展开综述,探讨其机制,期望为RNA干扰的应用研究提供一些思考。     

RNAi技术的医疗应用潜力

RNA干扰是指双链RNA在细胞内特异性地诱导同源互补的mRNA降解,从而阻断相应基因表达的现象。RNAi发展成为一种新型的基因治疗方式的进程取决于哺乳动物砌RNAi的研究进展。在大多数哺乳动物细胞中,直接导入长dsRNA引发的非特异性基因沉默掩盖了RNAi效应,而多种有效的双链RNA导入方式在一定程度上解决了这一问题。初步的实验结果表明,用RNAi治疗癌症、病毒感染等疾病的设想有可能变成现实。     

RNA干扰与技术

RNA干扰(RNA interference,RNAi)是由双链RNA诱导的、序列特异的基因沉默机制。它是自然存在于植物、线虫和果蝇中抵抗外来基因(包括病毒、转座子)入侵的方式。在哺乳动物细胞中,能够人工诱导RNA干扰,沉默有同源序列基因表达。这一新技术具有特异性、高效性。因此,正被用来研究人类基因组的功能、肿瘤和抗病毒感染等。     

RNA干扰在疾病治疗方面的应用研究

RNA干扰是由双链RNA引起的序列特异的基因沉默现象。由于RNA干扰能在细胞组织及动物模型中沉默疾病相关基因,因此,RNA干扰也是各种疾病治疗的有效手段。在哺乳动物细胞内诱导RNA干扰可以通过导入小干扰RNA（siRNA）,或是以质粒、病毒为载体表达短的发夹RNA（shRNA）而实现。本文介绍了RNA干扰在疾病治疗方面的应用,并就其面临的挑战进行讨论。     

RNA干扰技术在哺乳动物中的应用

RNA干扰(RNAi)是生物界普遍存在的一种抵御外来基因和病毒感染的进化保守机制.RNAi是由双链RNA触发的转录后基因沉默机制,具有序列特异性,在哺乳动物细胞中,RNAi由21～23个核苷酸组成的双链RNA引发.小干扰RNA(siRNA)可以在体外合成或通过表达载体在哺乳动物细胞内合成.由于RNAi技术具有快速、简单和特异性强等特点,在基因功能研究、抗病毒治疗和抗肿瘤治疗等方面有广泛的应用前景.     

RNA干扰技术及其在肝纤维化中的应用

RNA干扰(RNA interference,RNAi)是近几年发展起来的新技术,是外源和内源性双链RNA在生物体内诱导同源靶基因的mRNA特异性降解,因而抑制相应基因表达,导致转录后基因沉默的现象.尽管RNA干扰发现的时间较短,但由于其具有操作简单、成本低、特异性高和高效性等特点,因而发展迅速.小干扰RNA(small interfering RNA,siRNA)的可制备使RNAi在很多领域有了应用的前景,尤其是在复杂多变的肝脏疾病中.肝纤维化(hepatic fibrosis,HF)是多种慢性肝病发展的共同病理基础,RNAi技术在其基因治疗领域拥有广阔的前景.RNAi具有能够调节细胞增殖、抑制致病基因的表达、影响细胞的信号转导等方面的作用,可能成为肝纤维化有效的潜在治疗手段.     

siRNA及其在哺乳动物中的应用

RNA干扰现象已经在多种生物中发现,但是在多数哺乳动物中尚未发现自然存在RNA干扰的证据。因此最初RNA干扰技术在哺乳动物细胞中的应用受到很大的限制。直到对RNA干扰作用机制有了较深入的了解以后,主要是小干扰RNA的发现使RNA干扰技术在哺乳动物中的应用得以推广。本文介绍了RNA干扰,重点描述了小干扰RNA的发现、特点、现有制备方法以及应用。     

RNA干扰在植物中的作用机理及其应用研究进展

RNA干扰(RNAi)是广泛存在于生物中的一种现象,它是小干扰RNA诱导的转录后基因沉默,是生物抵抗异常DNA的一种保护机制,同时在生物生长发育过程中调控基因的表达.本文综述了近年来有关RNA干扰的发现、作用过程及其机理,分析了它与反义寡核苷酸、核酶、脱氧核酶的小同,并介绍了RNA干扰在植物基因功能、植物抗病毒、作物品种改良等方面的应用,为siRNA干扰的进一步利用提供参考资料.     

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific fashion.Small interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells.Here,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant tissue.Our results indicate that transiently expressed shRNA efficientlyinterfered with TMV infection.The interference observed is sequence-specific,and time-and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated tobamovirus.In order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation.Ourresults support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.     

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.     

Inhibition of virus replication by RNA interference

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene function by reverse genetics. Additionally, inhibition of virus replication by means of induced RNAi has now been reported for numerous viruses, including several important human pathogens such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, dengue virus, poliovirus and influenza virus A. In this review, we will summarize the current data on RNAi-mediated inhibition of virus replication and discuss the possibilities for the development of RNAi-based antiviral therapeutics.     

The response of mammalian cells to double-stranded RNA

Double-stranded RNA (dsRNA) has long been recognized as a central component of the interferon (IFN) system. It was originally characterized as a key mediator of IFN induction in response to virus infection. Subsequently, it was identified as a prime activator of the antiviral response. In recent years the discovery of the RNA interference (RNAi) pathway in mammals has renewed interest in dsRNA-mediated cellular responses. This has coincided with the identification of key components of the IFN induction pathway. Here, we present an overview of the current knowledge of dsRNA-mediated pathways in mammalian cells and introduce a link between these pathways and application of RNAi.     

RNAi技术及在植物抗病毒研究中的应用

RNA干扰(RNA interference,RNAi)技术是一项基因沉默新技术,在抗病毒研究中,人为地将与病毒或宿主基因(宿主基因编码的蛋白质对病毒很重要而对宿主本身作用很小或不起作用)同源的双链RNA(double strand RNA,dsRNA)导入生物体内,引起与其同源的基因发生沉默,从而抑制病毒复制,达到抗病毒的目的。因此RNAi技术在抗病毒研究中倍受关注,并取得了显著成绩。主要对RNAi技术的相关知识以及在植物抗病毒中的应用进展作一综述。     

RNAi的抗病毒研究进展

RNA干扰(RNA interference,RNAi)是真核生物中的特异核苷酸序列产生的基因沉默现象,被认为有抑制病毒复制的功能。最近的研究表明,通过诱导RNAi可以抑制多种病毒的复制,包括人类免疫缺陷病毒Ⅰ型,乙型肝炎病毒,丙型肝炎病毒,登革热病毒,脊髓灰质炎病毒,流感病毒,口蹄疫病毒和重症急性呼吸综合征病毒等。总结了目前运用RNA干扰技术抑制病毒复制的研究进展,展望基于RNAi技术的抗病毒治疗的可能性。     

Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2

RNA interference (RNAi) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. Viruses have also gained counter‐defensive measures by producing virulence determinants called viral‐suppressors‐of‐RNAi (VSRs). Interestingly, in spite of dominance of interferon‐based immunity over RNAi in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of RNAi in mammalian cells. The present study focuses on the modulation of intracellular RNAi during infection with rotavirus (RV), an enteric virus with double‐stranded RNA genome. Intriguingly, a time point‐dependent bimodal regulation of RNAi was observed in RV‐infected cells, where short interfering RNA (siRNA)‐based RNAi was rendered non‐functional during early hours of infection only to be reinstated fully beyond that early infection stage. Subsequent investigations revealed RV nonstructural protein 1 to serve as a putative VSR by associating with and triggering degradation of Argonaute2 (AGO2), the prime effector of siRNA‐mediated RNAi, via ubiquitin–proteasome pathway. The proviral significance of AGO2 degradation was further confirmed when ectopic overexpression of AGO2 significantly reduced RV infection. Cumulatively, the current study presents a unique modulation of host RNAi during RV infection, highlighting the importance of antiviral RNAi in mammalian cells.     

RNA干扰抑制乙型肝炎病毒复制及基因表达研究进展

RNA干扰(RNAinterference,RNAi)是指由21～23个核苷酸组成的双链RNA(dsRNA)所引发的生物细胞内同源基因转录后沉默的现象,是生物体在进化过程中普遍存在的一种基因调控机制。目前对由乙型肝炎病毒(HBV)引起的病毒性肝炎尚无令人满意的治疗效果,而RNA干扰技术的出现为各类慢性HBV感染的治疗开辟了新的途径。本文对RNA干扰抑制HBV复制及基因表达的研究现状、存在问题及应用前景进行了综述。