RNAi对基质金属蛋白酶13在兔骨性关节炎中表达的抑制作用

目的 观察RNA干扰技术(RNAi)是否能有效抑制兔OA模型膝关节软骨中基质金属蛋白酶13(MMP13)的表达.方法 体外化学合成MMP13序列特异性双链RNA与脂质体混合后转染OA模型中软骨细胞,实验分四组,特异性RNAi组:特异性RNAi+Lipofectamine2000,非特异RNAi组:非特异RNAi+Lipofectamine2000,脂质体组:Lipofectamine2000,对照组:无血清DMEM.用逆转录-聚合酶链反应(RT-PCR)和免疫组织化学法检测软骨细胞中的MMP13的表达.噻唑蓝(MTT)比色法检测各组的软骨细胞生长情况.结果 RT-PCR显示RNAi组较对照组、非特异性组和脂质体组的MMP13基因的PCR扩增条带明显减弱,免疫组织化学法显示RNAi组中MMP13阳性细胞数比对照组明显减少.与对照组比较,非特异性组、RNAi组、脂质体组软骨细胞的存活率显著降低(P<0.05),但在3组间两两差异无统计学意义.结论 使用RNA干扰技术可有效抑制兔OA模型中膝关节软骨细胞MMP13的表达,RNA干扰技术为基因治疗OA提供了新策略.     

RNA干扰与抗肝纤维化治疗前景

RNA干扰(RNA interference,RNAi)是近年来兴起的一项新技术,它可高效阻抑靶基因表达,不仅可用于研究基因功能,更是一种高效、特异的治疗手段.现就RNAi现象的发现、RNAi机制、意义及抗肝纤维化治疗前景作一简介. 　　……     

RNAi在膀胱癌研究中的应用

RNA干扰(RNA interference,RNAi)是正常生物体内抑制特定基因表达的一种现象,即当细胞中导入与内源性mRNA编码区同源的双链RNA时,该mRNA发生降解而导致基因表达沉默.近来随着RNAi的机制不断被阐明,其作为一种工具在肿瘤的发病机制及治疗方面的应用也日益广泛,本文就RNAi在膀胱癌研究中的进展作一综述.     

RNAa与肿瘤

小分子RNA在基因表达调控方面起着至关重要的作用.10年前研究发现小分子双链RNA(dsRNA)进入细胞后能导致同源性基因表达沉寂,即RNA干扰现象(RNAi).RNAi是由与靶基因具有同源性的dsRNA诱发的,这些dsRNA可产生于细胞内,由RNA酶Dicer将长的双链RNA分子切割成21个碱基对片断而生成;也可以在细胞外人工合成,然后引入细胞.     

RNA干扰应用减少肺缺血/再灌注损伤的研究进展

背景 肺移植中可产生缺血/再灌注损伤(ischemia/reperfusion injury,I/RI),主要以非特异性肺泡损伤、肺水肿、增加的肺血管阻力和缺氧为特征,并最终致移植物功能延迟恢复和移植器官失功. 目的 RNA干扰(RNA interfering,RNAi)基因沉默的进程可通过小分子干扰RNA(small interfering RNA,siRNA)开发出来,从而介导靶向细胞功能的精确控制,为改善肺I/RI提供新思路. 内容 回顾RNAi的发现及相关机制,同时对RNAi技术在肺I/RI模型的应用作一综述,并简介其应用于其他器官I/RI模型靶向治疗的案例. 趋向 基于RNAi的治疗学是一项新型且很有前景的策略,其在改善器官移植的I/RI动物模型的应用将愈将成熟,未来RNAi技术将会成功应用于肺移植的临床治疗.     

RNA干扰技术在肿瘤基因治疗方面的研究进展

RNA干扰技术(RNA interference,RNAi)在哺乳动物细胞中是利用小干扰RNA(small interference RNA,siRNA)诱导特异性基因表达抑制。其具有基因抑制效果确切、抑制具有严格的序列特异性、作用迅速等特点,故与基因替代、反义寡核苷酸治疗、细胞因子基因治疗等传统的肿瘤基因治疗方法相比,RNAi技术有着无可比拟的优势。本文对RNAi技术的作用机制及在肿瘤基因治疗方面的实验研究进展作一综述。     

RNA干扰与肿瘤治疗的研究进展

RNA干扰(RNA interference，RNAi)指与内源性mRNA编码区某段序列同源的双链RNA(double—stranded RNA，dsRNA)导入细胞时，该序列mRNA发生特异性的降解，并导致基因表达的沉默。小干扰RNA(small—imerfering RNA，siRNA)是RNAi作用的主要介质。其可经体外合成再转入细胞，也可通过各种载体内源性表达。RNAi在基因功能研究选择上具有独特的价值，在疾病的治疗上也有良好的应用前景。     

RNA干扰与肿瘤治疗的研究进展

RNA干扰 (RNAinterference ,RNAi)指与内源性mRNA编码区某段序列同源的双链RNA(double -strandedRNA ,dsRNA)导入细胞时 ,该序列mRNA发生特异性的降解 ,并导致基因表达的沉默。小干扰RNA(small-interferingRNA ,siRNA)是RNAi作用的主要介质。其可经体外合成再转入细胞 ,也可通过各种载体内源性表达。RNAi在基因功能研究选择上具有独特的价值 ,在疾病的治疗上也有良好的应用前景。     

RNAi基因沉默MCP-1在颈动脉粥样硬化形成中的影响

目的 探讨MCP-1小干扰RNA(siRNA)转染兔颈动脉粥样硬化模型对局部MCP-1及动脉粥样硬化的影响.方法 用大白兔建立颈动脉粥样硬化模型,模型分为3组:RNAi组12只,AS模型组8只,空质粒组8只.观察3组血管壁中泡沫细胞浸润及内膜/中膜厚度(I:M)比值及局部MCP-1表达情况.结果 MCP-1 siRNA转染后,发现RNAi组的I:M(1.46±0.23)较AS模型组(5.55±0.30)和空质粒组(5.27±0.31)显著降低(P<0.01);MCP-1 si RNA表达载体转染使局部MCP-1表达降低;RNAi组的m RNA表达水平较AS模型组和空质粒组下降.结论 MCP-1 siRNA转染抑制了局部MCP-1表达,延缓了动脉粥样硬化的发展,减轻了病变程度.     

RNA干扰技术及其在男科学研究中的应用

RNA干扰(RNA interference,RNAi),是一种在动植物中存在的通过双链RNA诱导同源特异性序列转录后基因沉默的过程。RNAi在抵抗病毒感染、抑制转座子活动、调控内源性基因表达等方面发挥着重要作用,已成为当前生物医学的研究热点之一。近年来,RNAi作为一种研究工具,已开始应用于ED、PCa及精子发生等男科学研究中,并取得了一定进展。本文就RNAi的基本原理、研究进展及其在男科学研究中的应用作一概述。     

RNA interference therapeutics in organ transplantation: The dawn of a new era

RNA interference (RNAi) is a natural process through which double‐stranded RNA molecules can silence the gene carrying the same code as the particular RNA of interest. In 2006, the discovery of RNAi was awarded the Nobel Prize in Medicine and its success has accumulated since. Gene silencing through RNAi has been used successfully in a broad range of diseases, and, more recently, this technique has gained interest in the field of organ transplantation. Here, genes related to ischemia‐reperfusion injury (IRI) or graft rejection may be silenced to improve organ quality after transplantation. Several strategies have been used to deliver siRNA, and pretransplant machine perfusion presents a unique opportunity to deliver siRNA to the target organ during ex situ preservation. In this review, the potential of RNAi in the field of organ transplantation will be discussed. A brief overview on the discovery of RNAi, its mechanism, and limitations are included. In addition, studies using RNAi to target genes related to IRI in liver, kidney, lung, and heart transplantation are discussed.     

RNA interference: a practical approach

BACKGROUND: Few new molecular biology techniques have advanced to find practical application as rapidly as RNA interference (RNAi). RNAi denotes the highly specific posttranslational silencing of gene expression that occurs in response to the introduction of double-stranded RNA into a cell. The purpose of this review is to present practical guidelines for designing and executing RNAi experiments. MATERIALS AND METHODS: Review of recent literature. RESULT AND CONCLUSION: We summarize the mechanisms underlying RNAi in mammalian cells and focus on practical advice for investigators conducting RNAi experiments. We suggest criteria to help select a suitable target gene sequence, define the structural characteristics of effective siRNAs, discuss transfection strategies, and describe experimental design, including important control methods. RNAi represents a powerful tool for determining the functions of specific genes.     

RNAi 在大肠癌基因治疗中的应用策略和展望

目的 探讨RNA干扰（RNA interference，RNAi）技术在大肠癌基因治疗研究中的应用。方法复习近几年的相关文献并进行综述。结果RNAi能够高效特异地沉默同源基因表达，可在大肠癌发生、发展多个环节发挥重要作用。结论RNAi技术为大肠癌基因治疗研究开辟了新的途径。     

Induction of accommodation model by combined RNA interference targeting 1,3-galactosyltransferase gene and low-dose GS-IB4 lectin in vitro

OBJECTIVE: This study sought to mimic the interaction of xenograft endothelial cells and human serum in vitro after successfully silencing the expression of porcine alpha1,3-galactosyltransferase (alpha1,3GT) gene by RNA interference (RNAi), and to investigate the possibility of inducing accommodation in vitro by stimulation of alpha-Gal-specific binding lectin, Griffonia simplicifolia isolectin B4 (GS-IB4) and RNAi. MATERIALS AND METHODS: Various alpha-Gal expression patterns on a pig endothelial cell immortalized line (PED) was achieved by serial doses of small interfering RNA (siRNA) targeting porcinc alpha1,3GT gene. alpha1,3GT-siRNA transfected PEDs were exposed to increasing doses of GS-IB4 lectin (0.5, 2, and 8 microg/mL) for 4 hours before incubation with normal human serum (NHS). Accommodation phenomenon of PEDs in NHS was observed by 51Cr release and antibody/complement binding assays. RESULTS: With combined RNAi and low-dose GS-IB4 stimulation, PEDs remarkably inhibited complement-mediated cytotoxicity, which showed a better protective effect than using RNAi alone. At a concentration of 2 mug/mL, GS-IB4 exhibited the maximum protective effect. The expression of E-selectin on alpha1,3GT-siRNA transfected PEDs did not differ from that on parental PEDs with heat-inactivated NHS (HINHS) stimulation. Combined with GS-IB4 stimulation, however, it inhibited expression of E-selectin, which was GS-IB4 dose dependent, resulting in mean fluorescence intensity values of 98.5, 42.0, and 36.3 at 0.5, 2, and 8 microg/mL. The mRNA expression of the protective gene HO-1 was significantly up-regulated after treatment with RNAi and low-dose of GS-IB4. CONCLUSIONS: Combined RNAi and low-dose GS-IB4 induced pig endothelial cell accommodation in vitro. The level of alpha-Gal expression played an important role in the induction of accommodation.     

人CCL5基因RNA干扰慢病毒载体的构建

目的 构建人CCL5基因RNA干扰(RNAi)慢病毒载体.方法 根据人CCL5基因信息,构建4个携带RNAi序列的pGCSIL-GFP质粒,与pHelper 1.0、Helper 2.0质粒一起利用293T细胞进行慢病毒包装.用CCL5 RNAi慢病毒载体感染人宫颈癌细胞(Hela),使用RT-PCR方法验证其干扰效率.结果 4个靶点中有3个靶点(a1、a2、a3)在Hela细胞上对CCL5基因的表达都有非常显著的敲减效果,敲减效率均达到95%以上.结论 构建的CCL5 RNAi慢病毒载体在Hela细胞中有较高的敲减效率,提示RNAi技术能够使细胞CCL5基因沉默.     

Sustained long-term RNA interference in nucleus pulposus cells in vivo mediated by unmodified small interfering RNA

RNA interference (RNAi) technology has recently emerged as an important biological strategy for gene silencing. Previously, the efficacies of RNAi in cultured nucleus pulposus cells in vitro have been reported. However, RNAi in the disc in vivo has never been reported. Therefore, the aims of the present study were to establish a method for RNAi in the disc in vivo and to evaluate the applicability of this technique for endogenous genes in the intervertebral discs using Fas Ligand (FasL) as a representative endogenous gene. To evaluate the efficacy of RNAi in vivo, two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids and unmodified short interference RNA (siRNA) duplex for targeting Firefly luciferase were co-transfected into coccygeal intervertebral disc of Sprague-Dawley rats in vivo using the ultrasound gene transfer technique. To evaluate the RNAi of the endogenous gene in vivo, siRNAs targeting rat FasL were transfected with the same technique. Non-specific siRNA was used as the negative control. The discs receiving no siRNAs were used as the control. The inhibitory effect of Firefly luciferase against Renilla luciferase was obtained using the results of dual-luciferase assay. Down-regulation of endogenous FasL was calculated by the data from real-time PCR. Our results showed that siRNA for Firefly luciferase can dramatically down-regulate the Firefly luciferase gene expression in vivo compared with Renilla luciferase. The inhibitory effects were maintained for at least 24 weeks and at 24 weeks post transfection, the inhibitory rate was 80% compared with the control group. Furthermore, the siRNA co-transfection group inhibited endogenous FasL expression by 53% compared with the control group. The present study demonstrates long-term down-regulation mediated by unmodified siRNA is possible not only for the exogenous reporter gene, but also for endogenous FasL expression in rat discs in vivo. This application of RNAi might be promising as a local therapy for disc degeneration and associated disorders by down-regulating some of the genes that are harmful for the normal physiology of the disc and may cause disc degeneration.     

RNA Interference: A Potent Tool for Gene-Specific Therapeutics

RNA interference (RNAi) is a process through which double-stranded RNA induces the activation of cellular pathways, leading to potent and selective silencing of genes with homology to the double strand. Much excitement surrounding small interfering RNA (siRNA)-mediated therapeutics arises from the fact that this approach overcomes many of the shortcomings previously experienced with approaches such as antibodies, antisense oligonucleotides and pharmacological inhibitors. Induction of RNAi through administration of siRNA has been successfully used in treatment of hepatitis, viral infections, and cancer. In this review we will present a brief history of RNAi, methods of inducing RNAi, application of RNAi in the therapeutic setting, and the possibilities of using this highly promising approach in the context of transplantation.