线粒体加工肽酶干扰RNA真核表达载体的构建

目的利用干扰RNA技术构建线粒体加工肽酶(mitochondrial processing peptidases,MPPs)干扰RNA真核表达载体,探讨MPPs在神经细胞中的作用,为帕金森病的研究提供有价值的资料。方法(1)根据Gen-Bank提供的大鼠MPPsα亚基(peptidase mitochondrial processing alpha,Pmpca)序列,设计并合成4对MPPs干扰RNA单链寡核苷酸:MPPs-SR-1113,MPPs-SR-1425,MPPs-SR-561,MPPs-SR-903。(2)使互补的单链寡核苷酸模板退火,再转化为能表达其小发卡结构RNA的DNA序列。(3)使PGPU6/GFP/Neo载体的线性化。(4)shRNA与载体的连接。(5)酶切鉴定并测序鉴定结果。结果(1)酶切结果表明,所有质粒均为阳性重组载体。(2)经鉴定证实,构建的siRNAs序列与基因库中序列完全相同,并且未发现有突变、缺失、插入等异常存在。结论利用干扰RNA技术可成功构建MPPs干扰RNA真核表达载体。     

线粒体加工肽酶干扰RNA真核表达载体转染PC12细胞系的构建及鉴定

目的利用干扰RNA技术构建线粒体加工肽酶(MPPs)的干扰RNA真核表达载体,稳定转染大鼠PC12细胞来抑制MPPs基因的表达,为研究MPPs蛋白的功能提供实验基础。方法脂质体转染已经构建好的4个MPPs干扰RNA真核表达载体和阴性对照干扰RNA真核表达载体到大鼠PC12细胞株,G418筛选阳性转染细胞克隆,制备稳定转染5个MPPs干扰RNA真核表达载体的PC12细胞系。用RT-PCR检测MPPs基因表达。结果获得稳定转染5个MPPs干扰RNA真核表达载体的PC12细胞系,RT-PCR证实所有4个干扰组(siRNA1,siRNA2,siRNA3 and siRNA4)MPPs-mRNA表达均下调,分别达到28%、38%、26%、16%。结论成功建立稳定转染MPPs干扰RNA的PC12细胞系。通过RT-PCR等相关方法证实MPPs干扰RNA真核表达载体转染PC12细胞成功。     

微小RNA和小干扰RNA在SCA3/MJD的研究进展

脊髓小脑共济失调3型(SCA3)为多聚谷氨酰胺(PolyQ)病,其致病基因为编码区内含胞嘧啶-腺嘌呤-鸟嘌呤(CAG)重复序列的ATXN3基因,编码蛋白ataxin-3羧基末端含PolyQ肽链,PolyQ扩展突变型ataxin-3蛋白可选择性地在中枢神经系统特定区域(小脑、脑干、脊髓等)聚积形成神经元核内包涵体.微小RNA是广泛存在于高等真核细胞中、长度为21 ～ 23 bp的小分子非编码RNA,与靶基因mRNA的3’端非编码区特异性结合后抑制靶基因的表达.小干扰RNA是RNA干扰技术中的效应分子,其长度为21～23 bp,特异性与mRNA的编码序列完全互补结合后,导致mRNA降解和沉默.二者在结构和作用机制存在许多类似之处,可用来进行基因功能分析和疾病发病机制的研究.     

RNAi技术及其在胶质瘤中的研究进展

<正>RNA干扰(RNA interference,RNAi)是一种在动植物中广泛存在的,由双链RNA(double-stranded RNA,dsRNA)介导的序列特异性基因沉默现象。由于RNA干扰可以抑制特定基因的表达,至今短短十几年的时间,RNAi技术已迅速发展成为一种成熟的基因沉默工具,对生命科学研究产生了深远的影响。已广泛用于研究基因表达及调控,信号传导通路,药物的作用机制,肿瘤的临床治疗等诸多领域,现将RNAi技术及其在胶质瘤研究中的应用进展综述如下。     

RNAi及其基因治疗研究进展

RNAi(RNA inter ference)即RNA干扰,是外源双链RNA(double-stranded RNA,dsRNA)分子在mRNA水平关闭相应序列基因表达所致的细胞内有效、特异性基因封闭,是广泛存在于生物界的古老现象,是生物体抵御病毒或其它外来核酸入侵以及保持自身遗传稳定的保护性机制,以其高效率,高特异性等特性,较反义核酸、核酶、三链RNA等基因沉默技术显示了独特优势及广泛的应用前景,本文就其特点、分子机制及其在基因治疗等方面的探索进行综述.     

小分子干扰RNA介导RhoA沉默优化骨髓间充质干细胞的培养

以往骨髓间充质干细胞的培养方法存在衰老和分化率低等问题。 目的：检测是否可以通过沉默RhoA基因的方法优化骨髓间充质干细胞培养。 方法：体外培养SD大鼠骨髓间充质干细胞,经小分子干扰RNA转染以沉默RhoA基因表达,分为3组培养：干细胞组(未转染小分子干扰RNA)、经随机打乱顺序的小分子干扰RNA 转染干细胞组、经小分子干扰RNA 转染的干细胞组。用RT-PCR,Western blot检测骨髓间充质干细胞在转染前后RhoA基因和蛋白的表达。应用细胞生长曲线、MTT比色法观察细胞生长的优化作用,采用流式细胞术测定细胞周期分布的变化。 结果与结论：与干细胞组、经随机打乱顺序的小分子干扰RNA 转染干细胞组比较,经小分子干扰RNA 转染的干细胞组RhoA基因和蛋白表达量明显降低(P < 0.05),细胞的生长速度明显增快,细胞周期G0/G1期减少,S期细胞数增多(P < 0.05)。说明通过沉默RhoA基因的方法可以促进骨髓间充质干细胞增殖,优化培养方法。     

RNA干涉技术在胶质瘤基因治疗研究中的进展

RNA干涉(RNA interfernce,RNAi)机理为利用双链RNA(dsRNA)特异性地降解相应序列的mRNA成为siRNA,从而特异性地阻断靶基因的表达,最近对其在应用于胶质瘤基因治疗方面的研究也有进展。本文介绍了RNA干扰的分子机制、技术应用及在胶质瘤基因治疗方面的进展和广阔的发展前景。     

NA干扰及其在神经变性性疾病研究中的应用

RNA干扰 (RNAi)是指生物体内利用具有同源性的双链RNA(dsRNA)诱发序列特异的转录后基因沉默(PTGS)的现象 ,它可以通过抑制蛋白表达模拟基因敲除技术。RNAi主要通过dsRNA被核酸酶Dicer切割成 2 1～ 2 5nt的小干扰RNA(siRNA) ,由siRNA介导识别并靶向切割同源mRNA分子而实现。随着研究的不断深入 ,RNAi的作用机制将逐步被阐明 ,其技术也将日趋完善和成熟 ,并将得到广泛的应用。本文就RNAi技术的研究进展及其在阿尔茨海默病、帕金森病等神经变性性疾病研究中的应用作一综述     

RNA干扰及其在神经变性性疾病研究中的应用

RNA干扰(RNAi)是指生物体内利用具有同源性的双链RNA(dsRNA)诱发序列特异的转录后基因沉默(PTGS)的现象，它可以通过抑制蛋白表达模拟基因敲除技术。RNAi主要通过dsRNA被核酸酶Dicer切割成21～25nt的小干扰RNA(siRNA)，由siRNA介导识别并靶向切割同源mRNA分子而实现。随着研究的不断深入，RNAi的作用机制将逐步被阐明，其技术也将日趋完善和成熟，并将得到广泛的应用。本文就RNAi技术的研究进展及其在阿尔茨海默病、帕金森病等神经变性性疾病研究中的应用作一综述。     

MicroRNA在胶质瘤形成及靶向治疗中的作用

Micro RNA(mi RNA)即小分子非编码RNA作为内源性干扰RNA的代表,能够显著改变靶蛋白表达,其在脑组织生理功能及神经母细胞瘤(GBM)发生、发展中的作用已引起广泛关注,并引起针对其治疗潜力及放化疗增敏方面的大量研究。本综述主要针对mi RNA在胶质瘤发生中的多重作用以及对病人的生存预后可能存在的临床意义进行讨论,并进一步阐述所选mi RNA在GBM以及其他中枢神经系统肿瘤临床研究中,作为治疗靶位的潜在应用价值。     

RNA interference and nonviral targeted gene therapy of experimental brain cancer

The human epidermal growth factor receptor (EGFR) plays an oncogenic role in solid cancer, including brain primary and metastatic cancers. Transvascular nonviral gene therapy in combination with EGFR-RNA interference (RNAi) represents a new therapeutic approach to silencing oncogenic genes in solid cancers. This is achieved with pegylated immunoliposomes (PIL) carrying short hairpin RNA expression plasmids driven by the U6 RNA polymerase promoter and directed to target EGFR expression by RNAi. The PIL is comprised of a mixture of known lipids containing polyethyleneglycol (PEG), which stabilizes the PIL structurein vivo in circulation. The tissue target specificity of PILs is given by conjugation of ∼1% of the PEG residues to monoclonal antibodies (mAbs) that bind to specific endogenous receptors (i.e., insulin and transferrin receptors) located in the brain vascular endothelium, which forms the blood brain barrier (BBB), and brain cellular membranes, respectively. These mAbs are known to induce 1) receptor-mediated transcytosis of the PIL complex through the BBB and 2) transport to the brain cell nuclear compartment. Treatment of an experimental human brain tumor model in scid mice is possible with weekly intravenous RNAi gene therapy causing reduced tumor expression of EGFR and 88% increase in survival time of these mice with advanced intracranial brain cancer. The availability of additional RNAi tumor targets may improve the therapeutic efficacy of this new anticancer drug. The accessibility to chimeric and/or humanized mAbs directed to human BBB and brain cell specific-receptors may accelerate the application of this technology to the treatment of human tumors.     

The long processes of short interfering RNAs--RNA interference and its implications in neuronal cells

Reverse genetics has been greatly advanced by the discovery of RNA interference (RNAi). This intracellular RNA-mediated gene silencing pathway is partially conserved from plants to mammals and offers a new powerful tool for the analysis of gene function. We give a brief overview of the discovery of RNAi, the underlying mechanisms and probable intrinsic roles of the pathway. Recent reports utilizing RNAi for gene silencing approaches in neuronal cells are reviewed and possible delivery techniques for small interfering RNA/double-stranded RNA are discussed.     

Developmental role of GABAB(1) receptors in Drosophila

Previously, an RNA interference (RNAi) knockdown of GABAB(1) subunit in adult Drosophila was used for behavioral studies. Here we report on developmental deficits caused by embryonic Drosophila GABAB(1) RNAi and drug antagonism. Injecting embryos with CGP54626 (a GABAB receptor antagonist) reduced hatching and caused lethality. Similar effects were produced by injecting embryos with GABAB(1) double-stranded RNA (RNAi). The surviving GABAB(1) RNAi larvae were significantly smaller than controls and showed a peculiar phenotype; their tracheae were folded. Our results suggest that GABAB receptors are required for normal development and that the Drosophila model could be used to investigate the participating molecular mechanisms.     

RNA interference in neuroscience

One of the most exciting findings in recent years has been the discovery of RNA interference (RNAi). RNAi is an important system that allows sequence-specific gene silencing through targeted degradation of mRNA by cognate double-stranded RNA (dsRNA). RNAi plays a role in endogenous cellular processes, such as developmental control and heterochromatin formation, and serves as an antiviral defense mechanism. Recent findings suggest that RNAi and related pathways are involved in protecting the genome against instabilities caused by transposons and repetitive sequences. Several rapidly developing RNAi methodologies provide a new approach for elucidation of gene functions. RNAi technology is also currently being evaluated as a potentially useful method to develop highly specific dsRNA-based gene-silencing therapeutics. In this paper, we review the use of RNAi in neuroscience research and as a possible therapeutic tool for treatment of neurological diseases.     

Promises and challenges in developing RNAi as a research tool and therapy for neurodegenerative diseases

RNA interference (RNAi) is a recently discovered mechanism that is conserved in a wide range of eukaryotic species. Triggered by double-stranded RNA, RNAi identifies and destroys the mRNA that shares homology with the double-stranded RNA. Because of its specificity, RNAi has a high potential for being a powerful investigative and therapeutic tool. Indeed, its use as a reverse genetics tool to determine gene functions in invertebrates and cultured mammalian cells has already been experiencing an explosive growth. Gratifyingly we have also seen its application in dissecting neurodegeneration pathways in vitro. Although early studies suggested that RNAi could be readily adapted for in vivo studies in mammals using the transgenic technology, difficulties including low transgenicity and low RNAi efficacy have emerged, which has prevented the wide use of transgenic RNAi. The potential of RNAi therapy for human diseases has been a great source of excitement. Several new studies have demonstrated this concept in animal models of neurodegenerative disease. In this review, we highlight the recent literature and our own data in applying RNAi in research and therapy in the area of neurodegenerative diseases. We discuss the present and future challenges in the full realization of the potential for RNAi.     

RNA干扰技术及其在神经科学研究中的应用

RNA干扰(RNAi)是指生物体内由双链RNA介导同源序列mRNA的特异性降解，从而导致基因沉默的现象。近年来，随着对RNAi研究的不断深入，其作用机制正在逐步被阐明；同时作为阻断基因表达的新手段，RNAi技术也日趋完善和成熟。以其高效性和特异性，RNAi为反向遗传学在神经科学研究中的应用提供了有力的工具。本文仅就RNAi技术的有关研究进展及其在神经科学研究中的应用作一概述。     

RNA interference technologies for understanding and treating neurodegenerative diseases

RNA interference (RNAi) is an evolutionarily conserved process that silences gene expression through double-stranded RNA species in a sequence-specific manner. With the completion of genome sequencing in multiple organisms, RNAi provides an efficient reverse genetics tool to reveal gene functions on a genome-wide scale. Conditional/inducible RNAi offers a new way to analyze gene function at different developmental stages and to create a new generation of animal models of human diseases. The sequence-specificity of RNAi and the fact that it is a naturally occurring process in human make it an excellent therapeutic tool for a wide range of diseases. This article provides a brief review of the current understandings of the mechanism of RNAi and its application to the nervous system, with particular focus on its application to understand mechanisms of neurodegenerative diseases. The prospects of the application of RNAi in clinical setting to treat these devastating diseases will also be presented.     

半乳糖凝集素-3对垂体腺瘤细胞增殖和凋亡的影响

目的观察半乳糖凝集素-3(Gal-3)对垂体腺瘤细胞生长的影响并探讨其可能的作用机制。方法采用RNA干扰(RNAi)抑制Gal-3基因的表达,应用Western blot分别检测Gal-3蛋白和细胞凋亡相关基因Bcl-2、Bax蛋白的表达情况,然后应用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑(MTT)法和流式细胞仪(FCM)检测大鼠垂体腺瘤细胞系GH3细胞增殖和细胞凋亡的情况。结果①RNAi后24 h、48 h,Gal-3均明显下调(P0.05),至72 h后较48 h略有回升(P0.05),同时伴有Bcl-2表达水平的明显下调(P0.05),而Bax蛋白表达无明显变化(P0.05);②RNAi下调Gal-3表达水平后,在不同时间点(24 h、48 h、72 h)GH3细胞的增殖能力均受到显著抑制(P0.05);③RNAi后GH3细胞的凋亡细胞比例明显增加,由处理前的5.2%上升至21.7%。结论下调Gal-3的表达导致促凋亡基因Bcl-2的表达下调,而抑凋亡基因Bax的表达水平不变,同时导致GH3细胞增殖受抑,细胞凋亡增加,提示Gal-3在垂体腺瘤细胞的增殖和进展中发挥重要作用,有望为垂体腺瘤的防治提供一个新的分子靶点。